Epigenetic Mechanisms Associated Leukemia

**13**

**Chapter 2**

Therapeutics

of the disease and treatment plans.

glucocorticoid, epigenetic drugs

response, and find novel targeted therapies.

**1. Epigenetics overview**

*and Duohui Jing*

**Abstract**

Epigenetic Landscape in Leukemia

and Its Impact on Antileukemia

*Bingzhi He, Julia Cathryn Hlavka-Zhang, Richard B. Lock* 

Epigenomic landscape mapping in leukemia cells supports germ line mutation studies to understand pathogenicity and treatment plans. The differential regulation of gene expression and heterogeneity between cell types during hematopoiesis and leukemia development is important in understanding oncogenesis. Oncogenesis in leukemia occurs at both genomic and epigenomic levels in order for hematological cells to evade lineage commitment. To ensure that therapies target the entire malignancy, it is important to consider the regulatory network that drives malignancy caused by mutations. Therapies tailored to respond to a patient-specific epigenetic landscape have the potential to minimize risk in administering chemotherapies that may not work. In this chapter, a focused study on childhood acute lymphoblastic leukemia (ALL) will be used as an example of the current research in the field of epigenetics in leukemia and the impact it carries on our understanding

**Keywords:** epigenome, childhood acute lymphoblastic leukemia, chemotherapy,

Epigenetics is a biological system that describes phenotypes and occurs due to differential regulation of genes (as opposed to genetic mutations). Current epigenetic studies include the control of basic biological functions [1], developmental biology [2, 3], the origin of disease [4], and cancer therapeutics [5]. Investigating the regulatory mechanisms driving these results involves genome-wide mapping of chromatin accessibility and conformation, transcription factor (TF) binding, DNA methylation, and histone modifications. Leukemia oncogenesis is a result of both genetic and epigenetic factors, whereby hematopoietic pathways are disrupted and leukemia cells are able to evade lineage commitment. This interdependence between altered genes and gene regulation is critical in cancer development. Consequently, a broader understanding inclusive of genomics and epigenomics will allow for development of drug targets which may limit cancer progression, improve therapeutic

#### **Chapter 2**

## Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics

*Bingzhi He, Julia Cathryn Hlavka-Zhang, Richard B. Lock and Duohui Jing*

#### **Abstract**

Epigenomic landscape mapping in leukemia cells supports germ line mutation studies to understand pathogenicity and treatment plans. The differential regulation of gene expression and heterogeneity between cell types during hematopoiesis and leukemia development is important in understanding oncogenesis. Oncogenesis in leukemia occurs at both genomic and epigenomic levels in order for hematological cells to evade lineage commitment. To ensure that therapies target the entire malignancy, it is important to consider the regulatory network that drives malignancy caused by mutations. Therapies tailored to respond to a patient-specific epigenetic landscape have the potential to minimize risk in administering chemotherapies that may not work. In this chapter, a focused study on childhood acute lymphoblastic leukemia (ALL) will be used as an example of the current research in the field of epigenetics in leukemia and the impact it carries on our understanding of the disease and treatment plans.

**Keywords:** epigenome, childhood acute lymphoblastic leukemia, chemotherapy, glucocorticoid, epigenetic drugs

#### **1. Epigenetics overview**

Epigenetics is a biological system that describes phenotypes and occurs due to differential regulation of genes (as opposed to genetic mutations). Current epigenetic studies include the control of basic biological functions [1], developmental biology [2, 3], the origin of disease [4], and cancer therapeutics [5]. Investigating the regulatory mechanisms driving these results involves genome-wide mapping of chromatin accessibility and conformation, transcription factor (TF) binding, DNA methylation, and histone modifications. Leukemia oncogenesis is a result of both genetic and epigenetic factors, whereby hematopoietic pathways are disrupted and leukemia cells are able to evade lineage commitment. This interdependence between altered genes and gene regulation is critical in cancer development. Consequently, a broader understanding inclusive of genomics and epigenomics will allow for development of drug targets which may limit cancer progression, improve therapeutic response, and find novel targeted therapies.

#### **1.1 Regulation of gene expression**

Multicellular eukaryotic organisms consist of a complex variety of different cells, all containing identical genomic DNA. The ability to create such diversity from identical duplicates of DNA is attributed to differential gene expression regulation. Gene expression is as product of epigenetic regulatory systems. Differences within these regulations result in differential expressions and subsequently, cell types that differ in structure and function [6].

The three-dimensional organization of DNA is central to gene expression, as it depends on physical access to the gene to be expressed. Genomic DNA is condensed and wrapped around histone proteins, forming chromatin [7]. Condensed chromatin structures inhibit gene transcription by making the gene physically inaccessible for transcriptional machinery to access (**Figure 1**). To unfold chromatin structure and expose the gene for transcription, endogenous mechanisms and drugs modify the histone proteins around which the DNA is bound [8, 9]. Modifications which result in gene silencing include histone deacetylation and DNA methylation. To achieve differential expression profiles between cell types, cells have different gene access profiles controlled by protein mediation, external stimuli, and development of cells [10].

#### *1.1.1 Histone deacetylation*

Histone deacetylation involves the switching of histone proteins from a positive to neutral charge via the addition of an acetyl to a lysine residue within the histone tail. This change in charge will result in the DNA (negatively charged) separation from the histone due to repulsion forces of like charges. Separated DNA regions become accessible to transcriptional machinery as a result. When this mechanism is reversed and lysines are deacetylated, DNA is attracted back toward the protein, resulting in tight DNA wrapping, thus inaccessible for transcriptional machinery (**Figure 2**) [11]. The enzyme responsible for acetylation of histone proteins is called histone acetyltransferase (HAT), which opens the chromatin structure and allows transcription. Deacetylating enzymes which cause compacting of chromatin are called histone deacetylases (HDAC).

#### *1.1.2 DNA methylation*

DNA methylation is another mechanism of chromatin remodeling. CpG islands are target sites in the genome for methylation by the DNA methyltransferase

#### **Figure 1.**

*Chromatin accessibility. Chromatin accessibility modulates gene expression. Densely packed chromatin (left) is inaccessible, preventing transcription factors (TFs), CCCTC-binding factor (CTCF), and polymerases from binding and subsequent gene expression. Opened chromatin structure (right) can be accessed by TFs, CTCF, and polymerases bindings, resulting in ability for gene transcription. Opened and closed chromatin structures are regulated by the acetylation status of histone proteins. Acetylated histones provide an open chromatin structure, while deacetylated histones form a closed chromatin structure. Adapted from Shlyueva et al., 2014 [10].*

**15**

genetic level.

**Figure 2.**

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics*

(DNMT) enzyme. Of the CpG sites in gene promoter regions, 70% are primary targets for methylation. DNMT prevents gene transcription via physically preventing transcription factor binding and by methylating DNA. Methylated DNA binds to methyl-CpG-binding domain (MBD) proteins that in turn recruit proteins such as histone deacetylase and other chromatin-remodeling proteins. In this new environment, chromatin becomes compact and inactive, termed heterochromatin

*Hierarchical layers of chromatin organization in mammalian cells. Adapted from Aranda et al., 2015 [11].*

ALL population studies indicated a trend in disease peak around the age of 5, after which there is no increase in prevalence (**Figure 3**). Evidences suggest hematopoietic regulatory network probably most highly involved in leukemia development at their highest in children <5 years old. Case-control studies have shown that the occurrence of childhood ALL is inversely linked to the degree of exposure to infections in the first few months of life [12, 13]. This suggests that there may be certain oncogenic factors present in the early days of a child's life that lead to the development of ALL, rather than being present later on or in adulthood. Addressing these up- and downregulations of oncogenic factors in this critical stage of hematopoiesis will provide insight into pathogenesis and progression of ALL beyond the

Epigenetics is still in the early stages of investigation and translational clinical use. Primary testing in clinics focuses on cytogenic studies, categorizing disease based on genetic abnormalities and cell markers, and then treating the patient accordingly; screening gene expression to map out regulatory profiles of a tumor are less established. In acute lymphoblastic leukemia (ALL), subtypes are diagnosed based on cytogenic testing and testing for markers [14]. In vivo mouse studies, however, have indicated that in almost 75% of diagnosed cases, chromosomal changes alone are insufficient to induce ALL [15]. Investigation of

*DOI: http://dx.doi.org/10.5772/intechopen.84184*

(repressed domain in **Figure 2**).

*1.2.1 Oncogenesis driven by epigenetics (in ALL)*

**1.2 Epigenetics in leukemia**

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.84184*

**Figure 2.**

*Germ Line Mutations Associated Leukemia*

**1.1 Regulation of gene expression**

*1.1.1 Histone deacetylation*

called histone deacetylases (HDAC).

*1.1.2 DNA methylation*

types that differ in structure and function [6].

Multicellular eukaryotic organisms consist of a complex variety of different cells, all containing identical genomic DNA. The ability to create such diversity from identical duplicates of DNA is attributed to differential gene expression regulation. Gene expression is as product of epigenetic regulatory systems. Differences within these regulations result in differential expressions and subsequently, cell

The three-dimensional organization of DNA is central to gene expression, as it depends on physical access to the gene to be expressed. Genomic DNA is condensed and wrapped around histone proteins, forming chromatin [7]. Condensed chromatin structures inhibit gene transcription by making the gene physically inaccessible for transcriptional machinery to access (**Figure 1**). To unfold chromatin structure and expose the gene for transcription, endogenous mechanisms and drugs modify the histone proteins around which the DNA is bound [8, 9]. Modifications which result in gene silencing include histone deacetylation and DNA methylation. To achieve differential expression profiles between cell types, cells have different gene access profiles controlled by protein mediation, external stimuli, and development of cells [10].

Histone deacetylation involves the switching of histone proteins from a positive to neutral charge via the addition of an acetyl to a lysine residue within the histone tail. This change in charge will result in the DNA (negatively charged) separation from the histone due to repulsion forces of like charges. Separated DNA regions become accessible to transcriptional machinery as a result. When this mechanism is reversed and lysines are deacetylated, DNA is attracted back toward the protein, resulting in tight DNA wrapping, thus inaccessible for transcriptional machinery (**Figure 2**) [11]. The enzyme responsible for acetylation of histone proteins is called histone acetyltransferase (HAT), which opens the chromatin structure and allows transcription. Deacetylating enzymes which cause compacting of chromatin are

DNA methylation is another mechanism of chromatin remodeling. CpG islands

are target sites in the genome for methylation by the DNA methyltransferase

*Chromatin accessibility. Chromatin accessibility modulates gene expression. Densely packed chromatin (left) is inaccessible, preventing transcription factors (TFs), CCCTC-binding factor (CTCF), and polymerases from binding and subsequent gene expression. Opened chromatin structure (right) can be accessed by TFs, CTCF, and polymerases bindings, resulting in ability for gene transcription. Opened and closed chromatin structures are regulated by the acetylation status of histone proteins. Acetylated histones provide an open chromatin structure, while deacetylated histones form a closed chromatin structure. Adapted from Shlyueva et al., 2014 [10].*

**14**

**Figure 1.**

*Hierarchical layers of chromatin organization in mammalian cells. Adapted from Aranda et al., 2015 [11].*

(DNMT) enzyme. Of the CpG sites in gene promoter regions, 70% are primary targets for methylation. DNMT prevents gene transcription via physically preventing transcription factor binding and by methylating DNA. Methylated DNA binds to methyl-CpG-binding domain (MBD) proteins that in turn recruit proteins such as histone deacetylase and other chromatin-remodeling proteins. In this new environment, chromatin becomes compact and inactive, termed heterochromatin (repressed domain in **Figure 2**).

#### **1.2 Epigenetics in leukemia**

ALL population studies indicated a trend in disease peak around the age of 5, after which there is no increase in prevalence (**Figure 3**). Evidences suggest hematopoietic regulatory network probably most highly involved in leukemia development at their highest in children <5 years old. Case-control studies have shown that the occurrence of childhood ALL is inversely linked to the degree of exposure to infections in the first few months of life [12, 13]. This suggests that there may be certain oncogenic factors present in the early days of a child's life that lead to the development of ALL, rather than being present later on or in adulthood. Addressing these up- and downregulations of oncogenic factors in this critical stage of hematopoiesis will provide insight into pathogenesis and progression of ALL beyond the genetic level.

#### *1.2.1 Oncogenesis driven by epigenetics (in ALL)*

Epigenetics is still in the early stages of investigation and translational clinical use. Primary testing in clinics focuses on cytogenic studies, categorizing disease based on genetic abnormalities and cell markers, and then treating the patient accordingly; screening gene expression to map out regulatory profiles of a tumor are less established. In acute lymphoblastic leukemia (ALL), subtypes are diagnosed based on cytogenic testing and testing for markers [14]. In vivo mouse studies, however, have indicated that in almost 75% of diagnosed cases, chromosomal changes alone are insufficient to induce ALL [15]. Investigation of

#### **Figure 3.**

*Incidence of ALL per 100,000 populations in 2010. Source: Australian Institute of Health and Welfare (AIHW), 2014 [97].*

#### **Figure 4.**

*B-ALL development. Multiple mutations contribute to the development of ALL. Mutations in ALL predisposing genes, e.g., IKZF1, and initiating genes, e.G., ETV6-RUNX1, and MLL rearrangement will promote ALL development. Alterations in B-cell development genes, e.G., PAX5 and IKZF1, inhibit cell maturation, resulting in the accumulation of immature cells. This alone however is not enough to cause ALL pathogenesis. Cell cycle and lymphoid lineage regulatory gene expression must also be altered to promote its survival. Further alterations may induce chemoresistance. Adapted from Mullighan, 2013 [15].*

genetic alterations alongside epigenetics microarrays of gene expression suggested an association between mutations and altered regulation in gene expressions during hematopoiesis in both B-ALL and T-ALL [14, 15]. Prenatal lesions and

**17**

[21, 37, 38].

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics*

postnatal-acquired mutations have also shown impaired regulation and development of progenitor B cells or T cells [16–18]. Together, these studies suggest that it is not mutations alone that act as oncogenic drivers but also an altered gene regula-

Thus far, ALL oncogenic studies have reported prenatal genetic lesions and inherited genetic predisposition which neither can stand alone to account for the disease. Prenatal genetic lesions have been reported with an unknown pathogenesis [18], and only 5% of ALL patients reported with an inherited genetic predisposition such as Down's syndrome and Bloom's syndrome [17, 19]. **Figure 4** indicates multiple genetic lesions contributing to an altered regulatory network in healthy lymphoid development toward a pathogenic ALL pathway [20–23]. Incomplete evidence regarding prenatal genetic lesions in ALL supports research into the epigenetic regulatory system in the development of ALL. Prenatal genetic lesions suggest the preleukemic states. Despite studies suggesting preleukemic state in utero, other studies show that the development of ALL in monozygotic twins follows a different time course. The difference in postnatal disease progression despite an identical prenatal state suggests the role of epigenetics in ALL

Since drugs target distinct cell pathways relying on gene expression, access to target genes is crucial for the treatment to work. A common ALL chemotherapy regimen of glucocorticoids relies on activation of the glucocorticoid receptor (GR), which binds to glucocorticoid-response elements (GREs) in gene promoters, to induce expression of pro-apoptotic pathways [28]. Using a DNase hypersensitivity assay (DHA) to determine chromatin accessibility [29], the majority of GR binding to GREs were identified in open chromatin [8, 30]. Thus, glucocorticoid therapy is dependent on GRE accessibility which is defined by chromatin accessibility. Resistance and patient response to such drugs is thought to be dependent on their

ALL is a malignant disease in both adults and children, with mutations developing along the lymphoid lineage starting at the lymphoid progenitor cells. Normally, lymphoid cells have the potential to differentiate into B or T cells, which under oncogenic conditions give rise to either B-ALL or T-ALL [17, 21]. Hematopoietic stem cells (HSCs), in the bone marrow, are the origin of both lymphoid and myeloid lineages. The tight regulation of gene expression in HSCs determines the lineage pathway and development. During oncogenesis, molecular defects and abnormal genes regulation may alter the differentiation of HSC; these factors may also contribute to further alterations downstream in hematopoiesis [17, 31–36]. Alterations along the lymphoid lineage result in abnormal pre-lymphoid cells called lymphoblasts; these aggressively proliferate and gradually replace the normal hematopoietic cells in the bone marrow and blood. Accumulation of lymphoblasts results in immunity retardation due to the insufficient amounts of mature lymphoid cells. Patients thus become immunocompromised and prone to various infectious diseases normally fought off by the immune system's lymphocytes

*DOI: http://dx.doi.org/10.5772/intechopen.84184*

tory network.

manifestation [24–27].

gene accessibility profiles.

*2.1.1 Acute lymphoblastic leukemia (ALL)*

**2. Targeting epigenome of ALL in chemotherapy**

**2.1 Glucocorticoid-based chemotherapy: Focused study on ALL**

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.84184*

postnatal-acquired mutations have also shown impaired regulation and development of progenitor B cells or T cells [16–18]. Together, these studies suggest that it is not mutations alone that act as oncogenic drivers but also an altered gene regulatory network.

Thus far, ALL oncogenic studies have reported prenatal genetic lesions and inherited genetic predisposition which neither can stand alone to account for the disease. Prenatal genetic lesions have been reported with an unknown pathogenesis [18], and only 5% of ALL patients reported with an inherited genetic predisposition such as Down's syndrome and Bloom's syndrome [17, 19]. **Figure 4** indicates multiple genetic lesions contributing to an altered regulatory network in healthy lymphoid development toward a pathogenic ALL pathway [20–23]. Incomplete evidence regarding prenatal genetic lesions in ALL supports research into the epigenetic regulatory system in the development of ALL. Prenatal genetic lesions suggest the preleukemic states. Despite studies suggesting preleukemic state in utero, other studies show that the development of ALL in monozygotic twins follows a different time course. The difference in postnatal disease progression despite an identical prenatal state suggests the role of epigenetics in ALL manifestation [24–27].

#### **2. Targeting epigenome of ALL in chemotherapy**

Since drugs target distinct cell pathways relying on gene expression, access to target genes is crucial for the treatment to work. A common ALL chemotherapy regimen of glucocorticoids relies on activation of the glucocorticoid receptor (GR), which binds to glucocorticoid-response elements (GREs) in gene promoters, to induce expression of pro-apoptotic pathways [28]. Using a DNase hypersensitivity assay (DHA) to determine chromatin accessibility [29], the majority of GR binding to GREs were identified in open chromatin [8, 30]. Thus, glucocorticoid therapy is dependent on GRE accessibility which is defined by chromatin accessibility. Resistance and patient response to such drugs is thought to be dependent on their gene accessibility profiles.

#### **2.1 Glucocorticoid-based chemotherapy: Focused study on ALL**

#### *2.1.1 Acute lymphoblastic leukemia (ALL)*

ALL is a malignant disease in both adults and children, with mutations developing along the lymphoid lineage starting at the lymphoid progenitor cells. Normally, lymphoid cells have the potential to differentiate into B or T cells, which under oncogenic conditions give rise to either B-ALL or T-ALL [17, 21]. Hematopoietic stem cells (HSCs), in the bone marrow, are the origin of both lymphoid and myeloid lineages. The tight regulation of gene expression in HSCs determines the lineage pathway and development. During oncogenesis, molecular defects and abnormal genes regulation may alter the differentiation of HSC; these factors may also contribute to further alterations downstream in hematopoiesis [17, 31–36]. Alterations along the lymphoid lineage result in abnormal pre-lymphoid cells called lymphoblasts; these aggressively proliferate and gradually replace the normal hematopoietic cells in the bone marrow and blood. Accumulation of lymphoblasts results in immunity retardation due to the insufficient amounts of mature lymphoid cells. Patients thus become immunocompromised and prone to various infectious diseases normally fought off by the immune system's lymphocytes [21, 37, 38].

*Germ Line Mutations Associated Leukemia*

**16**

**Figure 4.**

**Figure 3.**

*(AIHW), 2014 [97].*

genetic alterations alongside epigenetics microarrays of gene expression suggested an association between mutations and altered regulation in gene expressions during hematopoiesis in both B-ALL and T-ALL [14, 15]. Prenatal lesions and

*B-ALL development. Multiple mutations contribute to the development of ALL. Mutations in ALL predisposing genes, e.g., IKZF1, and initiating genes, e.G., ETV6-RUNX1, and MLL rearrangement will promote ALL development. Alterations in B-cell development genes, e.G., PAX5 and IKZF1, inhibit cell maturation, resulting in the accumulation of immature cells. This alone however is not enough to cause ALL pathogenesis. Cell cycle and lymphoid lineage regulatory gene expression must also be altered to promote its* 

*Incidence of ALL per 100,000 populations in 2010. Source: Australian Institute of Health and Welfare* 

*survival. Further alterations may induce chemoresistance. Adapted from Mullighan, 2013 [15].*

#### *2.1.2 Glucocorticoids in the clinic*

Glucocorticoids are naturally occurring steroid hormones that are widely recognized for their anti-inflammatory and immunosuppressing activities [39–41]. In leukemia, glucocorticoids are able to induce apoptosis in lymphoid cells. As such, glucocorticoid drugs such as dexamethasone and prednisolone are used as part of multi-agent chemotherapy regimens treating hematological malignancies [42–44], including ALL, chronic lymphocytic leukemia, multiple myeloma, and lymphoma. Due to pro-apoptotic pathway activation, glucocorticoids have remained the pivot point in chemotherapy treatment to combat ALL for 50 years [38, 45].

Glucocorticoids play a role in all three phases of treatment phases. During the remission-induction phase, glucocorticoids make up a significant portion of drug when administered in combination with vincristine and asparaginase and/or anthracycline. This initial high glucocorticoid portion aims to relieve at least 99% of the leukemic burden; the patient's response is critical in determining the future course of treatment and determining chance of relapse and prognosis [46]. The following two chemotherapy phases are less intensive, involving re-administration of remission-induction drugs in addition to methotrexate and mercaptopurine [17]. To note, some patients do not need to be administered pulses of glucocorticoids in phases two and three of the therapy, due to patients' contraindications [47]. This three-phase glucocorticoid regimen has seen an increase in ALL 5-year survival rate from 73–90% in the past 20 years [48], yet there still exits a subset of ALL patients who are resistant to glucocorticoids, resulting in poor prognosis.

#### *2.1.3 Glucocorticoid mechanism of action*

Glucocorticoid mode of action involves the activation of specific cellular pathways specific in lymphoid cells to induce cell death. Depending on cell type, cell-specific chromatin conformation provides the structural framework for transcription factor (TF) binding to regulate gene transcription that determines the ability of a cell to activate a pathway [49–51]. A cell-type-specific conformation is generated [11, 52] with each type having approximately 70,000–100,000 accessible chromatin domains and a network of cell-type-specific binding of transcriptional regulators [10, 53, 54]. Glucocorticoids are able to target intracellular pathways by interacting with the glucocorticoid receptor (GR) in the cytoplasm [55, 56]. The complex then translocates into the nucleus and binds at accessible chromatin domains containing glucocorticoid-response elements (GREs) at proximal promoter regions and/or distal sites of a gene [57–59]. GR binding to GREs induces chromatin remodeling and activates gene transcription via recruitment of other transcription proteins [60, 61]. To keep gene transcription tightly regulated, GR binding is highly selective and predetermined by chromatin accessibility in different cell types [8, 62]. Currently, the GR-binding landscape in different cell subsets, as well as between glucocorticoid-sensitive and resistant leukemia subsets, is yet to be established. Understanding this epigenetic landscape is crucial in understanding patient relapse or chemoresistance. Preliminary studies have started this investigation in pediatric ALL, to understand the mechanism of drug resistance in B-ALL.

#### *2.1.4 Limitations to glucocorticoid treatment*

While the glucocorticoid induces apoptosis pathway is still unclear, it is a pro-apoptotic pathway exclusive to lymphoid cells despite widespread expressions of GR in most human tissues [63, 64]. Glucocorticoids are rarely efficacious in treating myeloid leukemia [65]. Due to this lymphoid-specific apoptosis pathway,

**19**

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics*

is critical in optimizing glucocorticoid-based therapies in the clinic.

it is hypothesized that glucocorticoid-sensitive cells have a distinguished chromatin structure allowing for specific GR binding at GREs that glucocorticoid-resistant lymphoids, myeloid cells, and other tissue cells do not have [8, 66-69]. Therefore, understanding the lymphocyte-specific mechanisms of glucocorticoid-induced apoptosis, as well as the development of resistance to this class of steroid hormones,

The actions of glucocorticoids are cell type specific [65–67], although the exact molecular basis for this differential function remained elusive. While certain signaling pathways resulting from ALL oncogenes appear to interfere with glucocorticoid actions resulting in resistance, epigenetic evidence suggests that in addition to genetic alterations, epigenetic factors contribute to resistance. For instance, inhibition of GR expression or its translocation to the nucleus in vitro and in vivo models via *BTG1* or *PTEN* loss can cause glucocorticoid resistance by [70, 71]; however, GR function is rarely blocked in resistant ALL PDXs [72]. Mutations in epigenetic regulators such as *KMT2D*, *CREBBP*, and *HDAC7*, in two of five resistant PDX models, could not account for abnormal epigenetic changes. Mutations in various signaling pathways [73–76] have been reported to impair glucocorticoid-induced apoptosis in ALL by downregulating the GR-activated pro-apoptotic gene, *BIM* expression. The importance to study beyond gene mutations, toward epigenetic mapping of GR

binding, will provide a deeper understanding into individual drug response.

**2.2 Epigenetic landscape shapes the response to glucocorticoids in leukemia**

Epigenetic drugs inhibit and manipulate different epigenetic regulators involved in histone remodeling. Drugs which target epigenetic regulators can open closed chromatin structures commonly found in chemotherapy-resistant patients.

Lymphocyte-specific enhancers associated with glucocorticoid-induced apoptosis were identified in cell-wide studies [5, 28]. Moreover, aberrations at these enhancers were observed in glucocorticoid-resistant ALL cells. Similarly, nonlymphoid cells also exhibited inaccessible chromatin at these enhancers, providing insights into the cell-type-specific actions of glucocorticoids. A link between epigenetic differences and cell-type-specific actions of glucocorticoids are essential in the treatment determining treatment approaches for lymphoid malignancies. Lymphocyte-specific epigenetic modifications pre-determine glucocorticoid resistance in ALL and may account for the lack of glucocorticoid sensitivity in other cell types. Recent findings suggest that in glucocorticoid-sensitive cells, GR cooperates with the structural protein, CTCF, at lymphocyte-specific regulatory domains to mediate the formation of a transcriptionally active DNA loop to trigger gene transcription, which can be inhibited by increased DNA methylation in glucocorticoid-resistant ALL. By using a comprehensive map of chromatin accessibility, CTCF binding, histone modifications, and DNA methylation in normal and malignant cell types, there is evidence of regulatory heterogeneity in the epigenome of different cell types. Azacytidine, a DNA demethylating drug that is routinely used in the clinic, could partially reverse these changes and restore glucocorticoidinduced gene expression and glucocorticoid sensitivity. This indicates that reversal of epigenetic changes may lead to improvements in the use of glucocorticoids for

*DOI: http://dx.doi.org/10.5772/intechopen.84184*

the management of lymphoid malignancies.

**3. Epigenetic drugs**

**3.1 What are epigenetic drugs?**

#### *Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.84184*

it is hypothesized that glucocorticoid-sensitive cells have a distinguished chromatin structure allowing for specific GR binding at GREs that glucocorticoid-resistant lymphoids, myeloid cells, and other tissue cells do not have [8, 66-69]. Therefore, understanding the lymphocyte-specific mechanisms of glucocorticoid-induced apoptosis, as well as the development of resistance to this class of steroid hormones, is critical in optimizing glucocorticoid-based therapies in the clinic.

The actions of glucocorticoids are cell type specific [65–67], although the exact molecular basis for this differential function remained elusive. While certain signaling pathways resulting from ALL oncogenes appear to interfere with glucocorticoid actions resulting in resistance, epigenetic evidence suggests that in addition to genetic alterations, epigenetic factors contribute to resistance. For instance, inhibition of GR expression or its translocation to the nucleus in vitro and in vivo models via *BTG1* or *PTEN* loss can cause glucocorticoid resistance by [70, 71]; however, GR function is rarely blocked in resistant ALL PDXs [72]. Mutations in epigenetic regulators such as *KMT2D*, *CREBBP*, and *HDAC7*, in two of five resistant PDX models, could not account for abnormal epigenetic changes. Mutations in various signaling pathways [73–76] have been reported to impair glucocorticoid-induced apoptosis in ALL by downregulating the GR-activated pro-apoptotic gene, *BIM* expression. The importance to study beyond gene mutations, toward epigenetic mapping of GR binding, will provide a deeper understanding into individual drug response.

#### **2.2 Epigenetic landscape shapes the response to glucocorticoids in leukemia**

Lymphocyte-specific enhancers associated with glucocorticoid-induced apoptosis were identified in cell-wide studies [5, 28]. Moreover, aberrations at these enhancers were observed in glucocorticoid-resistant ALL cells. Similarly, nonlymphoid cells also exhibited inaccessible chromatin at these enhancers, providing insights into the cell-type-specific actions of glucocorticoids. A link between epigenetic differences and cell-type-specific actions of glucocorticoids are essential in the treatment determining treatment approaches for lymphoid malignancies. Lymphocyte-specific epigenetic modifications pre-determine glucocorticoid resistance in ALL and may account for the lack of glucocorticoid sensitivity in other cell types. Recent findings suggest that in glucocorticoid-sensitive cells, GR cooperates with the structural protein, CTCF, at lymphocyte-specific regulatory domains to mediate the formation of a transcriptionally active DNA loop to trigger gene transcription, which can be inhibited by increased DNA methylation in glucocorticoid-resistant ALL. By using a comprehensive map of chromatin accessibility, CTCF binding, histone modifications, and DNA methylation in normal and malignant cell types, there is evidence of regulatory heterogeneity in the epigenome of different cell types. Azacytidine, a DNA demethylating drug that is routinely used in the clinic, could partially reverse these changes and restore glucocorticoidinduced gene expression and glucocorticoid sensitivity. This indicates that reversal of epigenetic changes may lead to improvements in the use of glucocorticoids for the management of lymphoid malignancies.

#### **3. Epigenetic drugs**

#### **3.1 What are epigenetic drugs?**

Epigenetic drugs inhibit and manipulate different epigenetic regulators involved in histone remodeling. Drugs which target epigenetic regulators can open closed chromatin structures commonly found in chemotherapy-resistant patients.

*Germ Line Mutations Associated Leukemia*

Glucocorticoids are naturally occurring steroid hormones that are widely recognized for their anti-inflammatory and immunosuppressing activities [39–41]. In leukemia, glucocorticoids are able to induce apoptosis in lymphoid cells. As such, glucocorticoid drugs such as dexamethasone and prednisolone are used as part of multi-agent chemotherapy regimens treating hematological malignancies [42–44], including ALL, chronic lymphocytic leukemia, multiple myeloma, and lymphoma. Due to pro-apoptotic pathway activation, glucocorticoids have remained the pivot

Glucocorticoids play a role in all three phases of treatment phases. During the remission-induction phase, glucocorticoids make up a significant portion of drug when administered in combination with vincristine and asparaginase and/or anthracycline. This initial high glucocorticoid portion aims to relieve at least 99% of the leukemic burden; the patient's response is critical in determining the future course of treatment and determining chance of relapse and prognosis [46]. The following two chemotherapy phases are less intensive, involving re-administration of remission-induction drugs in addition to methotrexate and mercaptopurine [17]. To note, some patients do not need to be administered pulses of glucocorticoids in phases two and three of the therapy, due to patients' contraindications [47]. This three-phase glucocorticoid regimen has seen an increase in ALL 5-year survival rate from 73–90% in the past 20 years [48], yet there still exits a subset of ALL patients

Glucocorticoid mode of action involves the activation of specific cellular pathways specific in lymphoid cells to induce cell death. Depending on cell type, cell-specific chromatin conformation provides the structural framework for

While the glucocorticoid induces apoptosis pathway is still unclear, it is a pro-apoptotic pathway exclusive to lymphoid cells despite widespread expressions of GR in most human tissues [63, 64]. Glucocorticoids are rarely efficacious in treating myeloid leukemia [65]. Due to this lymphoid-specific apoptosis pathway,

transcription factor (TF) binding to regulate gene transcription that determines the ability of a cell to activate a pathway [49–51]. A cell-type-specific conformation is generated [11, 52] with each type having approximately 70,000–100,000 accessible chromatin domains and a network of cell-type-specific binding of transcriptional regulators [10, 53, 54]. Glucocorticoids are able to target intracellular pathways by interacting with the glucocorticoid receptor (GR) in the cytoplasm [55, 56]. The complex then translocates into the nucleus and binds at accessible chromatin domains containing glucocorticoid-response elements (GREs) at proximal promoter regions and/or distal sites of a gene [57–59]. GR binding to GREs induces chromatin remodeling and activates gene transcription via recruitment of other transcription proteins [60, 61]. To keep gene transcription tightly regulated, GR binding is highly selective and predetermined by chromatin accessibility in different cell types [8, 62]. Currently, the GR-binding landscape in different cell subsets, as well as between glucocorticoid-sensitive and resistant leukemia subsets, is yet to be established. Understanding this epigenetic landscape is crucial in understanding patient relapse or chemoresistance. Preliminary studies have started this investigation in pediatric ALL, to understand the mechanism of drug resistance in B-ALL.

point in chemotherapy treatment to combat ALL for 50 years [38, 45].

who are resistant to glucocorticoids, resulting in poor prognosis.

*2.1.3 Glucocorticoid mechanism of action*

*2.1.4 Limitations to glucocorticoid treatment*

*2.1.2 Glucocorticoids in the clinic*

**18**

**21**

**Figure 6.**

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics*

During preparation of genetic information in the S phase of the cell cycle, replication machinery is responsible for DNA replication, and DNMT functions duplicate methylation status by adding methyl groups to the DNA accordingly (**Figure 6**). DNMT inhibitors, azacytidine and decitabine, prevent DNMT methylation. Azacytidine and decitabine are metabolized inside cells into 5-aza-2′-deoxycytidine-triphosphate. The difference between DNMT bounding to 5-aza-2′-deoxycytidine-triphosphate and DNMT bound to cytosine is used to inhibit DNMT. As illustrated in **Figure 7**, DNMT reversibly binds with cytosine, which allows DNMT to be released from DNA through beta-elimination once methylation is completed. DNMT binding to 5-aza-2′-deoxycytidine-triphosphate establishes a covalent bond preventing beta-elimination; therefore, DNMT remains bond to the DNA. Subsequently, the error triggers DNA damage signaling and the trapped DNMT is degraded. As a result, methylation markers get lost during DNA replication [78]. Demethylated DNA allows for an open chromatin structure to be accessed by transcription factors induce by chemotherapeutic

Single-agent study conducted by Khaldoun Al-Romaih's group showed that decitabine therapy had a cytotoxic effect mediated by the removal of hypomethylation of the CpG position both in vivo and in vitro. The cells treated with decitabine showed significant cell death in vitro, and six pro-apoptotic genes (GADD45A, HSPA9B, PAWR, PDCD5, NFKBIA, and TNFAIP3) were induced to ≥ twofold in vivo [79]. Combination therapy trials revealed the value of DNMT inhibition in addition to current therapeutic regimens. In one clinical trial in refractory ALL patients, decitabine combined with hyper-CVAD (fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with high-dose methotrexate and cytarabine) was able to achieve complete remission in patients that did

*Effect of epigenetic modification on gene expression. Activation of gene transcription needs transcription factor binding to the promoter region of the gene. Without DNA methylation, transcription factors and RNA polymerase II (RNA pol II) can bind to DNA segments; however, when methyl group is added to the DNA by DNA methyltransferase (DNMT), the methylation not only impedes the binding of transcription factors to DNA but also recruits histone deacetylase (HDAC) causing chromatin structure to become compacted, which* 

*places spatial limitation for transcription factor binding [83].*

*DOI: http://dx.doi.org/10.5772/intechopen.84184*

*3.2.1 DNMT inhibitors*

drugs such as glucocorticoids.

#### **Figure 5.**

*Epigenetic reader, writer, and eraser. Epigenetic writers such as histone acetyltransferases (HATs), histone methyltransferases (HMTs), protein arginine methyltransferases (PRMTs), and kinases lay down epigenetic marks on amino acid residues on histone tails. Epigenetic readers are proteins that contain bromodomains, chromodomains, and Tudor domains, allowing them to bind to these epigenetic marks. Epigenetic erasers such as histone deacetylases, lysine demethylases, and phosphatases catalyze the removal of epigenetic marks. Together they modulate chromatin structures and regulate various DNA-dependent biological processes such as DNA synthesis and replication. Adapted from Falkenberg and Johnstone, 2014 [77].*

Epigenetic regulators can be divided into different categories based on their method of modification: epigenetic writers, readers, and erasers (**Figure 5**).

Epigenetic regulators determine gene expression, and an understanding of them allows for the development of drugs to regulate gene expression over epigenetic marks. Epigenetic writers lay down epigenetic marks on DNA or amino acid residues on histones tails [77]. Examples include histone acetyltransferases (HATs), histone methyltransferases (HMTs), and protein arginine methyltransferases (PRMTs). Drugs which target these are DNA hypomethylating agents, bromodomains, and HDAC inhibitors. Epigenetic readers are proteins that contain bromodomains, chromodomains, and Tudor domains allowing them to bind to specific epigenetic marks on chromatin. Epigenetic erasers such as histone deacetylases, lysine demethylases, and phosphatases catalyze the removal of epigenetic marks.

#### **3.2 Categories of epigenetic modifying drugs**

The epigenetic drugs to be discussed are designed to target different epigenetic regulators responsible for gene silencing.

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.84184*

#### *3.2.1 DNMT inhibitors*

*Germ Line Mutations Associated Leukemia*

Epigenetic regulators can be divided into different categories based on their method

The epigenetic drugs to be discussed are designed to target different epigenetic

Epigenetic regulators determine gene expression, and an understanding of them allows for the development of drugs to regulate gene expression over epigenetic marks. Epigenetic writers lay down epigenetic marks on DNA or amino acid residues on histones tails [77]. Examples include histone acetyltransferases (HATs), histone methyltransferases (HMTs), and protein arginine methyltransferases (PRMTs). Drugs which target these are DNA hypomethylating agents, bromodomains, and HDAC inhibitors. Epigenetic readers are proteins that contain bromodomains, chromodomains, and Tudor domains allowing them to bind to specific epigenetic marks on chromatin. Epigenetic erasers such as histone deacetylases, lysine demethylases, and phosphatases catalyze the removal of epigenetic marks.

*Epigenetic reader, writer, and eraser. Epigenetic writers such as histone acetyltransferases (HATs), histone methyltransferases (HMTs), protein arginine methyltransferases (PRMTs), and kinases lay down epigenetic marks on amino acid residues on histone tails. Epigenetic readers are proteins that contain bromodomains, chromodomains, and Tudor domains, allowing them to bind to these epigenetic marks. Epigenetic erasers such as histone deacetylases, lysine demethylases, and phosphatases catalyze the removal of epigenetic marks. Together they modulate chromatin structures and regulate various DNA-dependent biological processes such as* 

of modification: epigenetic writers, readers, and erasers (**Figure 5**).

*DNA synthesis and replication. Adapted from Falkenberg and Johnstone, 2014 [77].*

**3.2 Categories of epigenetic modifying drugs**

regulators responsible for gene silencing.

**20**

**Figure 5.**

During preparation of genetic information in the S phase of the cell cycle, replication machinery is responsible for DNA replication, and DNMT functions duplicate methylation status by adding methyl groups to the DNA accordingly (**Figure 6**). DNMT inhibitors, azacytidine and decitabine, prevent DNMT methylation. Azacytidine and decitabine are metabolized inside cells into 5-aza-2′-deoxycytidine-triphosphate. The difference between DNMT bounding to 5-aza-2′-deoxycytidine-triphosphate and DNMT bound to cytosine is used to inhibit DNMT. As illustrated in **Figure 7**, DNMT reversibly binds with cytosine, which allows DNMT to be released from DNA through beta-elimination once methylation is completed. DNMT binding to 5-aza-2′-deoxycytidine-triphosphate establishes a covalent bond preventing beta-elimination; therefore, DNMT remains bond to the DNA. Subsequently, the error triggers DNA damage signaling and the trapped DNMT is degraded. As a result, methylation markers get lost during DNA replication [78]. Demethylated DNA allows for an open chromatin structure to be accessed by transcription factors induce by chemotherapeutic drugs such as glucocorticoids.

Single-agent study conducted by Khaldoun Al-Romaih's group showed that decitabine therapy had a cytotoxic effect mediated by the removal of hypomethylation of the CpG position both in vivo and in vitro. The cells treated with decitabine showed significant cell death in vitro, and six pro-apoptotic genes (GADD45A, HSPA9B, PAWR, PDCD5, NFKBIA, and TNFAIP3) were induced to ≥ twofold in vivo [79]. Combination therapy trials revealed the value of DNMT inhibition in addition to current therapeutic regimens. In one clinical trial in refractory ALL patients, decitabine combined with hyper-CVAD (fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with high-dose methotrexate and cytarabine) was able to achieve complete remission in patients that did

#### **Figure 6.**

*Effect of epigenetic modification on gene expression. Activation of gene transcription needs transcription factor binding to the promoter region of the gene. Without DNA methylation, transcription factors and RNA polymerase II (RNA pol II) can bind to DNA segments; however, when methyl group is added to the DNA by DNA methyltransferase (DNMT), the methylation not only impedes the binding of transcription factors to DNA but also recruits histone deacetylase (HDAC) causing chromatin structure to become compacted, which places spatial limitation for transcription factor binding [83].*

#### **Figure 7.**

*Reversible binding of cytosine with DNMT in DNA methylation process versus irreversible binding of 5-azacytosine with DNMT when leukemia cells were treated with azacytidine, which leads to degradation of DNMT and subsequent loss of methylation. Adapted from Stresemann and Lyko, 2008 [78].*

not respond to hyper-CVAD treatment alone. Patient DNA analysis confirmed that the hypomethylation status in the combination treatment group was the reason for reversal of resistance [80]. A number of other clinical studies provided similar results, showing synergistic effects between hypomethylating agents and several chemotherapy agents. Agents such as prednisolone, etoposides, doxorubicin, and cytarabine were shown to increase chemosensitivity in leukemia cells [81–84]. Although promising not all experimental groups responded, indicating the need for further research into the complex network of interactions.

#### *3.2.2 HDAC inhibitors*

An open chromatin structure also relies on the relatively loose interactions between DNA and histone proteins. The addition of an acetyl groups to the lysine amino acids on histone proteins by histone acetyltransferases (HATs) reduces the positive charges on the histone proteins. DNA, negatively charged, is therefore less attracted to these now less positive histones, thus less tightly bound and easier for transcription factors to access [83]. Conversely, the removal of acetylation by histone deacetylase (HDAC) forms condensed heterochromatin and silences critical apoptosis gene transcriptions. HDAC inhibitors prevent this form of gene silencing and are used alongside standard chemotherapy to promote pro-apoptotic pathways.

HDAC enzyme family consists of Class I (HDAC1, HDAC2, HDAC3, HDAC8), Class II (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, HDAC10), Class III (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7), and Class IV (HDAC11). HDAC inhibitors are designed as either selective inhibitors or pan-inhibitors (against all types of HDAC). Evidence that some HDACs play a stronger role in cancer development and patient prognosis than others makes specific HDAC inhibitors more appealing for clinical use. A study of 94 pediatric ALL patients showed differential HDAC expressions between the T-ALL and B-ALL subtypes. For T-ALL, HDAC1 and HDAC4 showed a higher expression than in B-ALL. T-ALL patients with HDAC3 expression above the cohort median also displayed a significantly higher 5-year event-free survival (EFS). In B-ALL, HDAC5 had higher expression than

**23**

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics*

in T-ALL. In both T-cell and B-cell ALL, HDAC7 and HADC9 expression levels higher than the cohort median were associated with a lower 5-year EFS [85]. These trends suggest that Class II HDACs are associated with poorer prognosis; hence, a

Bromodomains (BRDs) are epigenetic readers which selectively bind to acetylate lysine of histones tails, regulating gene expression [86]. Bromodomain-containing proteins that target genes are primarily cell cycle M/G1 genes in mitotic chromatin (expressed at the end or immediately after mitosis); late-phase genes were not found to be BRD4 bound. M/G1 gene expression during telophase coincides with histone H3 and H4 acetylation in those genes. BRD binding to M/G1 genes was associated with recruitment of positive transcription elongation factor b (P-TEFb), resulting in translational memory in the daughter cells [87]. BRD binds to MYC and activates enhancer-binding protein 4 (AP4) promoters. AP4 is a key mediator of mitogenicity for proto-oncogene MYC [88]. Upon AP4 activation by MYC, repress-

JQ1 is a BRD inhibitor which acts on the MYC-AP4 axis [89]. Direct inhibition by JQ1 of BRD binding to the MYC and AP4 promoters indirectly results in cell cycle arrest as a *P21*-induced response to DNA damage P53 or TGFb/Smad signaling

Epigenetic drugs are useful in combination with cytotoxic drugs, due to their ability to allow for access to pro-apoptotic pathways, otherwise blocked by epigenetic silencing. In a subset of leukemia patients, resistance to cytotoxic drugs such as glucocorticoids and methotrexate is a result of inaction of pro-apoptotic genes. Theoretically, applying epigenetic drugs such as those mentioned above should remove the epigenetic modification such that pro-apoptotic genes may go from

Chromatin conformation and gene expression studies at the glucocorticoidinduced pro-apoptotic *BIM* gene in drug-sensitive versus resisted lymphoid cells indicated that glucocorticoid resistance in ALL patients may be due to epigenetic

Studies showed closed DNMT catalyzed chromatin structure caused by DNA methylation impedes the transcription of *BIM*; lymphocyte-specific open chromatin structure determines *BIM* expression. Therefore, modifying chromatin structure would allow for *BIM* expression. Common cytosine analog hypomethylating drugs, decitabine or azacytidine, used in ALL act by inhibiting DNMT activity thus reactivate silenced genes. Once metabolized inside cells into 5-aza-2′ deoxycytidine-triphosphate, cytosine substrates on DNA replication machinery are replaced by the drug analog. These DNMT inhibitors have been proven to work synergistically with glucocorticoids in glucocorticoid-resistant ALLs, increasing the

*DOI: http://dx.doi.org/10.5772/intechopen.84184*

*3.2.3 Bromodomain inhibitors*

ing cell cycle arrests gene *P21* [88].

**3.3 Epigenetic drugs in chemotherapy**

*3.3.1 Epigenetic drugs to treat glucocorticoid resistance*

overall effectiveness of the therapeutic regimens [84].

pathways [90].

repression to promotion.

modifications.

*3.3.2 DNMT inhibitors*

specific inhibition of this class of HDACs is important.

in T-ALL. In both T-cell and B-cell ALL, HDAC7 and HADC9 expression levels higher than the cohort median were associated with a lower 5-year EFS [85]. These trends suggest that Class II HDACs are associated with poorer prognosis; hence, a specific inhibition of this class of HDACs is important.

#### *3.2.3 Bromodomain inhibitors*

*Germ Line Mutations Associated Leukemia*

not respond to hyper-CVAD treatment alone. Patient DNA analysis confirmed that the hypomethylation status in the combination treatment group was the reason for reversal of resistance [80]. A number of other clinical studies provided similar results, showing synergistic effects between hypomethylating agents and several chemotherapy agents. Agents such as prednisolone, etoposides, doxorubicin, and cytarabine were shown to increase chemosensitivity in leukemia cells [81–84]. Although promising not all experimental groups responded, indicating the need for

*Reversible binding of cytosine with DNMT in DNA methylation process versus irreversible binding of 5-azacytosine with DNMT when leukemia cells were treated with azacytidine, which leads to degradation of* 

*DNMT and subsequent loss of methylation. Adapted from Stresemann and Lyko, 2008 [78].*

An open chromatin structure also relies on the relatively loose interactions between DNA and histone proteins. The addition of an acetyl groups to the lysine amino acids on histone proteins by histone acetyltransferases (HATs) reduces the positive charges on the histone proteins. DNA, negatively charged, is therefore less attracted to these now less positive histones, thus less tightly bound and easier for transcription factors to access [83]. Conversely, the removal of acetylation by histone deacetylase (HDAC) forms condensed heterochromatin and silences critical apoptosis gene transcriptions. HDAC inhibitors prevent this form of gene silencing and are used alongside standard chemotherapy to promote

HDAC enzyme family consists of Class I (HDAC1, HDAC2, HDAC3, HDAC8), Class II (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, HDAC10), Class III (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7), and Class IV (HDAC11). HDAC inhibitors are designed as either selective inhibitors or pan-inhibitors (against all types of HDAC). Evidence that some HDACs play a stronger role in cancer development and patient prognosis than others makes specific HDAC inhibitors more appealing for clinical use. A study of 94 pediatric ALL patients showed differential HDAC expressions between the T-ALL and B-ALL subtypes. For T-ALL, HDAC1 and HDAC4 showed a higher expression than in B-ALL. T-ALL patients with HDAC3 expression above the cohort median also displayed a significantly higher 5-year event-free survival (EFS). In B-ALL, HDAC5 had higher expression than

further research into the complex network of interactions.

*3.2.2 HDAC inhibitors*

**Figure 7.**

pro-apoptotic pathways.

**22**

Bromodomains (BRDs) are epigenetic readers which selectively bind to acetylate lysine of histones tails, regulating gene expression [86]. Bromodomain-containing proteins that target genes are primarily cell cycle M/G1 genes in mitotic chromatin (expressed at the end or immediately after mitosis); late-phase genes were not found to be BRD4 bound. M/G1 gene expression during telophase coincides with histone H3 and H4 acetylation in those genes. BRD binding to M/G1 genes was associated with recruitment of positive transcription elongation factor b (P-TEFb), resulting in translational memory in the daughter cells [87]. BRD binds to MYC and activates enhancer-binding protein 4 (AP4) promoters. AP4 is a key mediator of mitogenicity for proto-oncogene MYC [88]. Upon AP4 activation by MYC, repressing cell cycle arrests gene *P21* [88].

JQ1 is a BRD inhibitor which acts on the MYC-AP4 axis [89]. Direct inhibition by JQ1 of BRD binding to the MYC and AP4 promoters indirectly results in cell cycle arrest as a *P21*-induced response to DNA damage P53 or TGFb/Smad signaling pathways [90].

#### **3.3 Epigenetic drugs in chemotherapy**

Epigenetic drugs are useful in combination with cytotoxic drugs, due to their ability to allow for access to pro-apoptotic pathways, otherwise blocked by epigenetic silencing. In a subset of leukemia patients, resistance to cytotoxic drugs such as glucocorticoids and methotrexate is a result of inaction of pro-apoptotic genes. Theoretically, applying epigenetic drugs such as those mentioned above should remove the epigenetic modification such that pro-apoptotic genes may go from repression to promotion.

#### *3.3.1 Epigenetic drugs to treat glucocorticoid resistance*

Chromatin conformation and gene expression studies at the glucocorticoidinduced pro-apoptotic *BIM* gene in drug-sensitive versus resisted lymphoid cells indicated that glucocorticoid resistance in ALL patients may be due to epigenetic modifications.

#### *3.3.2 DNMT inhibitors*

Studies showed closed DNMT catalyzed chromatin structure caused by DNA methylation impedes the transcription of *BIM*; lymphocyte-specific open chromatin structure determines *BIM* expression. Therefore, modifying chromatin structure would allow for *BIM* expression. Common cytosine analog hypomethylating drugs, decitabine or azacytidine, used in ALL act by inhibiting DNMT activity thus reactivate silenced genes. Once metabolized inside cells into 5-aza-2′ deoxycytidine-triphosphate, cytosine substrates on DNA replication machinery are replaced by the drug analog. These DNMT inhibitors have been proven to work synergistically with glucocorticoids in glucocorticoid-resistant ALLs, increasing the overall effectiveness of the therapeutic regimens [84].

#### *3.3.3 HDAC inhibitors*

Suberoylanilide hydroxamic acid (SAHA; Vorinostat) is an HDAC inhibitor shown to work synergistically with different chemotherapies. Glucocorticoidresistant ALL cases were associated to a correlation between histone H3K9 deacetylation and pro-apoptotic gene, *BIM*, repression [91, 92]. SAHA acts to increase acetylation at H3K9 for *BIM* expression. Another chemocytotoxic pathway involves FPGS conversion of methotrexate to a cytotoxic product responsible for apoptosis, MTX-PGs. HDAC1 represses *FPGS* via epigenetically silencing. The combination of SAHA with methotrexate was shown to increase *FPGS* expression by two- to fivefolds, thus increasing cytotoxic activity [93].

#### **3.4 Combination therapy**

Combination therapy aims to bring synergistic effects by targeting both cell death pathway (chemotherapy, e.g., glucocorticoids) and access to this pathway (epigenetic drug). Of the clinical trials underway, the use of relatively high doses of 5-azacitidine (150 mg/m2 as a continuous infusion daily × 5 days) is combined with cytarabine in patients relapsed from cytarabine alone. It was hypothesized that treatment with 5-azacitidine could induce expression of deoxycytidine kinase. Two out of the 17 patients achieved complete responses (CR). In another study, decitabine was combined with either amsacrine or idarubicin in patients with acute leukemia. CR was achieved in 36% (23 out 63) of patients, with a median diseasefree survival of 8 months [82].

Using a hypomethylating agent such as decitabine, glucocorticoids in resistant ALL patients had the potential to expose the pro-apoptotic gene *BIM*, making it available for GR binding and subsequent transcription; thus reversing patient glucocorticoid resistance [5]. This should especially increase the rate of CR among the patients with glucocorticoid-resistant ALL and prolong event-free survival as suggested in preclinical trial model.

#### **4. Conclusion**

Knowledge of gene regulation has deepened the understanding of cellular mechanisms and disease development. In leukemia, genomic and epigenomic landscapes together provide crucial disease mechanism of pathogenicity and drug resistance [94–96]. Epigenetics is the driver of life and diversity of different organisms, and equally able to dysregulate cells and cause diseases such as leukemia. Hematopoiesis is a tightly controlled process essential for life, therefore, unless appropriately regulated, susceptible to regulatory errors as oncogenic drivers alongside mutations. Lineage-specific landscapes have been shown to be involved in hematopoiesis and leukemia evolution [51], providing a backbone for understanding targetable and non-targetable sites within different leukemic subtypes.

Studying a level of cell dysfunction preceding DNA mutations has allowed for understanding into pathogenesis and drug resistance which could not be correlated to DNA sequencing. Understanding resistance to chemotherapies, lowering patient prognosis, has been enlightened in epigenetic studies. For example, actions of glucocorticoids are cell type-specific and can be used in lymphocyte-specific leukemia cells to induce cell death [66, 67]. Analysis between genome-wide lymphocytespecific open chromatin domains (LSOs) and integrated LSOs with glucocorticoidinduced RNA transcription and chromatin modulation in ALL was performed to causes glucocorticoid resistance beyond gene mutations [5]. LSOs critical for

**25**

†

**Author details**

Bingzhi He†

Australia

provided the original work is properly cited.

, Julia Cathryn Hlavka-Zhang†

\*Address all correspondence to: djing@ccia.unsw.edu.au

Both authors contributed equally to this work

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Children's Cancer Institute, Lowy Cancer Research Centre, UNSW, Sydney, NSW,

, Richard B. Lock and Duohui Jing\*

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics*

glucocorticoid-induced apoptosis were identified as well as structural protein CTCF binding in this region. These findings showed that upon GR binding to the LSO and CTCF binding, DNA would loop at the pro-apoptotic *BIM* gene and could be expressed. Crucially, DNA methylation (closed chromatin structure) was present in glucocorticoid-resistant ALL and nonlymphoid cell types, preventing DNA looping

Understanding the importance of chromatin accessibility has allowed for identification of glucocorticoid sensitivity in cells and provides promising drug response predictions. Furthermore, development of epigenetic drugs that may modify chromatin to be accessible is currently being investigated. This would allow for more effective drug treatments to disrupt the oncogenesis driven via dysregulated

*DOI: http://dx.doi.org/10.5772/intechopen.84184*

and *BIM* expression.

pathways.

#### *Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.84184*

glucocorticoid-induced apoptosis were identified as well as structural protein CTCF binding in this region. These findings showed that upon GR binding to the LSO and CTCF binding, DNA would loop at the pro-apoptotic *BIM* gene and could be expressed. Crucially, DNA methylation (closed chromatin structure) was present in glucocorticoid-resistant ALL and nonlymphoid cell types, preventing DNA looping and *BIM* expression.

Understanding the importance of chromatin accessibility has allowed for identification of glucocorticoid sensitivity in cells and provides promising drug response predictions. Furthermore, development of epigenetic drugs that may modify chromatin to be accessible is currently being investigated. This would allow for more effective drug treatments to disrupt the oncogenesis driven via dysregulated pathways.

#### **Author details**

*Germ Line Mutations Associated Leukemia*

fivefolds, thus increasing cytotoxic activity [93].

Suberoylanilide hydroxamic acid (SAHA; Vorinostat) is an HDAC inhibitor shown to work synergistically with different chemotherapies. Glucocorticoidresistant ALL cases were associated to a correlation between histone H3K9 deacetylation and pro-apoptotic gene, *BIM*, repression [91, 92]. SAHA acts to increase acetylation at H3K9 for *BIM* expression. Another chemocytotoxic pathway involves FPGS conversion of methotrexate to a cytotoxic product responsible for apoptosis, MTX-PGs. HDAC1 represses *FPGS* via epigenetically silencing. The combination of SAHA with methotrexate was shown to increase *FPGS* expression by two- to

Combination therapy aims to bring synergistic effects by targeting both cell death pathway (chemotherapy, e.g., glucocorticoids) and access to this pathway (epigenetic drug). Of the clinical trials underway, the use of relatively high doses

with cytarabine in patients relapsed from cytarabine alone. It was hypothesized that treatment with 5-azacitidine could induce expression of deoxycytidine kinase. Two out of the 17 patients achieved complete responses (CR). In another study, decitabine was combined with either amsacrine or idarubicin in patients with acute leukemia. CR was achieved in 36% (23 out 63) of patients, with a median disease-

Using a hypomethylating agent such as decitabine, glucocorticoids in resistant ALL patients had the potential to expose the pro-apoptotic gene *BIM*, making it available for GR binding and subsequent transcription; thus reversing patient glucocorticoid resistance [5]. This should especially increase the rate of CR among the patients with glucocorticoid-resistant ALL and prolong event-free survival as

Knowledge of gene regulation has deepened the understanding of cellular mechanisms and disease development. In leukemia, genomic and epigenomic landscapes together provide crucial disease mechanism of pathogenicity and drug resistance [94–96]. Epigenetics is the driver of life and diversity of different organisms, and equally able to dysregulate cells and cause diseases such as leukemia. Hematopoiesis is a tightly controlled process essential for life, therefore, unless appropriately regulated, susceptible to regulatory errors as oncogenic drivers alongside mutations. Lineage-specific landscapes have been shown to be involved in hematopoiesis and leukemia evolution [51], providing a backbone for understanding targetable and

Studying a level of cell dysfunction preceding DNA mutations has allowed for understanding into pathogenesis and drug resistance which could not be correlated to DNA sequencing. Understanding resistance to chemotherapies, lowering patient prognosis, has been enlightened in epigenetic studies. For example, actions of glucocorticoids are cell type-specific and can be used in lymphocyte-specific leukemia cells to induce cell death [66, 67]. Analysis between genome-wide lymphocytespecific open chromatin domains (LSOs) and integrated LSOs with glucocorticoidinduced RNA transcription and chromatin modulation in ALL was performed to causes glucocorticoid resistance beyond gene mutations [5]. LSOs critical for

as a continuous infusion daily × 5 days) is combined

*3.3.3 HDAC inhibitors*

**3.4 Combination therapy**

of 5-azacitidine (150 mg/m2

free survival of 8 months [82].

suggested in preclinical trial model.

non-targetable sites within different leukemic subtypes.

**4. Conclusion**

**24**

Bingzhi He† , Julia Cathryn Hlavka-Zhang† , Richard B. Lock and Duohui Jing\* Children's Cancer Institute, Lowy Cancer Research Centre, UNSW, Sydney, NSW, Australia

\*Address all correspondence to: djing@ccia.unsw.edu.au

† Both authors contributed equally to this work

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Amin V et al. Epigenomic footprints across 111 reference epigenomes reveal tissue-specific epigenetic regulation of lincRNAs. Nature Communications. 2015;**6**:6370

[2] Ziller MJ et al. Dissecting neural differentiation regulatory networks through epigenetic footprinting. Nature. 2015;**518**(7539):355-359

[3] Dixon JR et al. Chromatin architecture reorganization during stem cell differentiation. Nature. 2015;**518**(7539):331-336

[4] De Jager PL et al. Alzheimer's disease: Early alterations in brain DNA methylation at ANK1, BIN1, RHBDF2 and other loci. Nature Neuroscience. 2014;**17**(9):1156-1163

[5] Jing D et al. Lymphocyte-specific chromatin accessibility pre-determines glucocorticoid resistance in acute lymphoblastic leukemia. Cancer Cell. 2018;**34**(6):906-921 e8

[6] Alvarez-Errico D et al. Epigenetic control of myeloid cell differentiation, identity and function. Nature Reviews. Immunology. 2015;**15**(1):7-17

[7] Luger K, Dechassa ML, Tremethick DJ. New insights into nucleosome and chromatin structure: An ordered state or a disordered affair? Nature Reviews. Molecular Cell Biology. 2012;**13**(7):436-447

[8] John S et al. Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nature Genetics. 2011;**43**(3):264-268

[9] Margueron R, Reinberg D. Chromatin structure and the inheritance of epigenetic information. Nature Reviews. Genetics. 2010;**11**(4):285-296

[10] Shlyueva D, Stampfel G, Stark A. Transcriptional enhancers: From properties to genome-wide predictions. Nature Reviews. Genetics. 2014;**15**(4):272-286

[11] Aranda S, Mas G, Di Croce L. Regulation of gene transcription by polycomb proteins. Science Advances. 2015;**1**(11):e1500737

[12] Wiemels J. Perspectives on the causes of childhood leukemia. Chemico-Biological Interactions. 2012;**196**(3):59-67

[13] Gilham C et al. Day care in infancy and risk of childhood acute lymphoblastic leukaemia: Findings from UK case-control study. BMJ. 2005;**330**(7503):1294

[14] Mullighan CG. The genomic landscape of acute lymphoblastic leukemia in children and young adults. Hematology. American Society of Hematology. Education Program. 2014;**2014**(1):174-180

[15] Mullighan CG. Genomic characterization of childhood acute lymphoblastic leukemia. Seminars in Hematology. 2013;**50**(4):314-324

[16] Dang J et al. PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia. Blood. 2015;**125**(23):3609-3617

[17] Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet. 2008;**371**(9617):1030-1043

[18] Roberts KG, Mullighan CG. Genomics in acute lymphoblastic leukaemia: Insights and treatment implications. Nature Reviews. Clinical Oncology. 2015;**12**(6):344-357

[19] Bercovich D et al. Mutations of JAK2 in acute lymphoblastic leukaemias

**27**

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics*

[29] Crawford GE et al. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Research.

[30] Wiench M et al. DNA methylation status predicts cell type-specific enhancer activity. The EMBO Journal.

[31] Schindler JW et al. TEL-AML1 corrupts hematopoietic stem cells to persist in the bone marrow and initiate leukemia. Cell Stem Cell.

[32] Zelent A, Greaves M, Enver T. Role of the TEL-AML1 fusion gene in the molecular pathogenesis of childhood acute lymphoblastic leukaemia. Oncogene. 2004;**23**(24):4275-4283

[33] Armstrong SA, Look AT. Molecular

leukemia. Journal of Clinical Oncology.

[34] Di Cello F et al. Inactivation of the Cdkn2a locus cooperates with HMGA1 to drive T-cell leukemogenesis.

[35] Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. The New England Journal of Medicine.

[36] Mullighan CG et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature.

[37] Speck NA, Gilliland DG. Corebinding factors in haematopoiesis and leukaemia. Nature Reviews Cancer.

[38] Torpy JM, Lynm C, Glass RM. JAMA patient page. Acute lymphoblastic leukemia. JAMA. 2009;**301**(4):452

genetics of acute lymphoblastic

2005;**23**(26):6306-6315

Leukemia & Lymphoma. 2013;**54**(8):1762-1768

2004;**350**(15):1535-1548

2007;**446**(7137):758-764

2002;**2**(7):502-513

2006;**16**(1):123-131

2011;**30**(15):3028-3039

2009;**5**(1):43-53

*DOI: http://dx.doi.org/10.5772/intechopen.84184*

[20] Levine RL. Inherited susceptibility to pediatric acute lymphoblastic leukemia. Nature Genetics.

JM. Pathogenesis and prognostication in acute lymphoblastic leukemia. F1000Prime Reports. 2014;**6**:59

[22] Hunger SP, Mullighan CG. Acute lymphoblastic leukemia in children. The New England Journal of Medicine.

[23] Swaminathan S et al. Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia. Nature Immunology. 2015;**16**(7):766-774

[24] Ford AM et al. Fetal origins of the TEL-AML1 fusion gene in identical twins with leukemia. Proceedings of the National Academy of Sciences of the United States of America.

[25] Wasserman R et al. Predominance

[26] Francis SS et al. Mode of delivery and risk of childhood leukemia. Cancer Epidemiology, Biomarkers & Prevention. 2014;**23**(5):876-881

[28] Jing D et al. Opposing regulation of BIM and BCL2 controls glucocorticoidinduced apoptosis of pediatric acute lymphoblastic leukemia cells. Blood.

associated with down's syndrome. Lancet. 2008;**372**(9648):1484-1492

2009;**41**(9):957-958

[21] Zuckerman T, Rowe

2015;**373**(16):1541-1552

1998;**95**(8):4584-4588

1992;**176**(6):1577-1581

[27] Copley MR, Eaves

2015;**125**(2):273-283

2013;**45**:e55

CJ. Developmental changes in hematopoietic stem cell properties. Experimental and Molecular Medicine.

of fetal type DJH joining in young children with B precursor lymphoblastic leukemia as evidence for an in utero transforming event. The Journal of Experimental Medicine. *Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.84184*

associated with down's syndrome. Lancet. 2008;**372**(9648):1484-1492

[20] Levine RL. Inherited susceptibility to pediatric acute lymphoblastic leukemia. Nature Genetics. 2009;**41**(9):957-958

[21] Zuckerman T, Rowe JM. Pathogenesis and prognostication in acute lymphoblastic leukemia. F1000Prime Reports. 2014;**6**:59

[22] Hunger SP, Mullighan CG. Acute lymphoblastic leukemia in children. The New England Journal of Medicine. 2015;**373**(16):1541-1552

[23] Swaminathan S et al. Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia. Nature Immunology. 2015;**16**(7):766-774

[24] Ford AM et al. Fetal origins of the TEL-AML1 fusion gene in identical twins with leukemia. Proceedings of the National Academy of Sciences of the United States of America. 1998;**95**(8):4584-4588

[25] Wasserman R et al. Predominance of fetal type DJH joining in young children with B precursor lymphoblastic leukemia as evidence for an in utero transforming event. The Journal of Experimental Medicine. 1992;**176**(6):1577-1581

[26] Francis SS et al. Mode of delivery and risk of childhood leukemia. Cancer Epidemiology, Biomarkers & Prevention. 2014;**23**(5):876-881

[27] Copley MR, Eaves CJ. Developmental changes in hematopoietic stem cell properties. Experimental and Molecular Medicine. 2013;**45**:e55

[28] Jing D et al. Opposing regulation of BIM and BCL2 controls glucocorticoidinduced apoptosis of pediatric acute lymphoblastic leukemia cells. Blood. 2015;**125**(2):273-283

[29] Crawford GE et al. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Research. 2006;**16**(1):123-131

[30] Wiench M et al. DNA methylation status predicts cell type-specific enhancer activity. The EMBO Journal. 2011;**30**(15):3028-3039

[31] Schindler JW et al. TEL-AML1 corrupts hematopoietic stem cells to persist in the bone marrow and initiate leukemia. Cell Stem Cell. 2009;**5**(1):43-53

[32] Zelent A, Greaves M, Enver T. Role of the TEL-AML1 fusion gene in the molecular pathogenesis of childhood acute lymphoblastic leukaemia. Oncogene. 2004;**23**(24):4275-4283

[33] Armstrong SA, Look AT. Molecular genetics of acute lymphoblastic leukemia. Journal of Clinical Oncology. 2005;**23**(26):6306-6315

[34] Di Cello F et al. Inactivation of the Cdkn2a locus cooperates with HMGA1 to drive T-cell leukemogenesis. Leukemia & Lymphoma. 2013;**54**(8):1762-1768

[35] Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. The New England Journal of Medicine. 2004;**350**(15):1535-1548

[36] Mullighan CG et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature. 2007;**446**(7137):758-764

[37] Speck NA, Gilliland DG. Corebinding factors in haematopoiesis and leukaemia. Nature Reviews Cancer. 2002;**2**(7):502-513

[38] Torpy JM, Lynm C, Glass RM. JAMA patient page. Acute lymphoblastic leukemia. JAMA. 2009;**301**(4):452

**26**

*Germ Line Mutations Associated Leukemia*

[1] Amin V et al. Epigenomic footprints across 111 reference epigenomes reveal tissue-specific epigenetic regulation of lincRNAs. Nature Communications.

[10] Shlyueva D, Stampfel G, Stark A. Transcriptional enhancers: From properties to genome-wide predictions. Nature Reviews. Genetics.

[11] Aranda S, Mas G, Di Croce L. Regulation of gene transcription by polycomb proteins. Science Advances.

[12] Wiemels J. Perspectives on the causes of childhood leukemia. Chemico-Biological Interactions.

[13] Gilham C et al. Day care in infancy and risk of childhood acute lymphoblastic leukaemia: Findings from UK case-control study. BMJ.

[14] Mullighan CG. The genomic landscape of acute lymphoblastic leukemia in children and young adults. Hematology. American Society of Hematology. Education Program.

2014;**15**(4):272-286

2015;**1**(11):e1500737

2012;**196**(3):59-67

2005;**330**(7503):1294

2014;**2014**(1):174-180

[15] Mullighan CG. Genomic characterization of childhood acute lymphoblastic leukemia. Seminars in Hematology. 2013;**50**(4):314-324

[16] Dang J et al. PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia.

Blood. 2015;**125**(23):3609-3617

2008;**371**(9617):1030-1043

[18] Roberts KG, Mullighan

Oncology. 2015;**12**(6):344-357

[19] Bercovich D et al. Mutations of JAK2 in acute lymphoblastic leukaemias

[17] Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet.

CG. Genomics in acute lymphoblastic leukaemia: Insights and treatment implications. Nature Reviews. Clinical

[2] Ziller MJ et al. Dissecting neural differentiation regulatory networks through epigenetic footprinting. Nature.

2015;**518**(7539):355-359

2015;**518**(7539):331-336

2014;**17**(9):1156-1163

2018;**34**(6):906-921 e8

2012;**13**(7):436-447

2011;**43**(3):264-268

2010;**11**(4):285-296

[9] Margueron R, Reinberg D. Chromatin structure and the inheritance of epigenetic information.

Nature Reviews. Genetics.

[3] Dixon JR et al. Chromatin architecture reorganization during stem cell differentiation. Nature.

[4] De Jager PL et al. Alzheimer's disease: Early alterations in brain DNA methylation at ANK1, BIN1, RHBDF2 and other loci. Nature Neuroscience.

[5] Jing D et al. Lymphocyte-specific chromatin accessibility pre-determines glucocorticoid resistance in acute lymphoblastic leukemia. Cancer Cell.

[6] Alvarez-Errico D et al. Epigenetic control of myeloid cell differentiation, identity and function. Nature Reviews.

[7] Luger K, Dechassa ML, Tremethick DJ. New insights into nucleosome and chromatin structure: An ordered state or a disordered affair? Nature Reviews. Molecular Cell Biology.

[8] John S et al. Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nature Genetics.

Immunology. 2015;**15**(1):7-17

2015;**6**:6370

**References**

[39] Goulding NJ, Flower RJ. Glucocorticoids, Milestones in drug therapy. Basel, Switzerland: Boston: Birkhäuser Verlag; 2001. p. 205

[40] Dalakas MC. Inflammatory muscle diseases. The New England Journal of Medicine. 2015;**372**(18):1734-1747

[41] Nair P et al. Oral glucocorticoidsparing effect of benralizumab in severe asthma. The New England Journal of Medicine. 2017;**376**(25):2448-2458

[42] Inaba H, Pui CH. Glucocorticoid use in acute lymphoblastic leukaemia. The Lancet Oncology. 2010;**11**(11):1096-1106

[43] Kim IK et al. Glucocorticoidinduced tumor necrosis factor receptor-related protein co-stimulation facilitates tumor regression by inducing IL-9-producing helper T cells. Nature Medicine. 2015;**21**(9):1010-1017

[44] Palumbo A et al. Daratumumab, bortezomib, and dexamethasone for multiple myeloma. The New England Journal of Medicine. 2016;**375**(8):754-766

[45] Pui CH, Evans WE. A 50-year journey to cure childhood acute lymphoblastic leukemia. Seminars in Hematology. 2013;**50**(3):185-196

[46] Klumper E et al. In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia. Blood. 1995;**86**(10):3861-3868

[47] Howard SC et al. Urolithiasis in pediatric patients with acute lymphoblastic leukemia. Leukemia. 2003;**17**(3):541-546

[48] Australian Institute of Health and Welfare. A Picture of Australia's Children 2012. Canberra: Australian Institute of Health and Welfare; 2012

[49] Guenther MG et al. A chromatin landmark and transcription initiation at most promoters in human cells. Cell. 2007;**130**(1):77-88

[50] Li G et al. Extensive promotercentered chromatin interactions provide a topological basis for transcription regulation. Cell. 2012;**148**(1-2):84-98

[51] Corces MR et al. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nature Genetics. 2016;**48**(10):1193-1203

[52] Hnisz D, Day DS, Young RA. Insulated neighborhoods: Structural and functional units of mammalian gene control. Cell. 2016;**167**(5):1188-1200

[53] Thurman RE et al. The accessible chromatin landscape of the human genome. Nature. 2012;**489**(7414):75-82

[54] Perera D et al. Differential DNA repair underlies mutation hotspots at active promoters in cancer genomes. Nature. 2016;**532**(7598):259-263

[55] Greenstein S et al. Mechanisms of glucocorticoid-mediated apoptosis in hematological malignancies. Clinical Cancer Research. 2002;**8**(6):1681-1694

[56] Watson LC et al. The glucocorticoid receptor dimer interface allosterically transmits sequence-specific DNA signals. Nature Structural & Molecular Biology. 2013;**20**(7):876-883

[57] John S et al. Interaction of the glucocorticoid receptor with the chromatin landscape. Molecular Cell. 2008;**29**(5):611-624

[58] Paakinaho V et al. Glucocorticoid receptor activates poised FKBP51 locus through long-distance interactions. Molecular Endocrinology. 2010;**24**(3):511-525

[59] Vockley CM et al. Direct GR binding sites potentiate clusters of TF

**29**

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics*

resistance in acute lymphoblastic leukemia. The Journal of Steroid Biochemistry and Molecular Biology.

[69] Schmidt S et al. Glucocorticoid resistance in two key models of acute lymphoblastic leukemia occurs at the level of the glucocorticoid receptor. The FASEB Journal. 2006;**20**(14):2600-2602

[70] Piovan E et al. Direct reversal of glucocorticoid resistance by AKT inhibition in acute

lymphoblastic leukemia. Cancer Cell.

[71] van Galen JC et al. BTG1 regulates glucocorticoid receptor autoinduction in acute lymphoblastic leukemia. Blood.

[72] Bachmann PS et al. Divergent mechanisms of glucocorticoid resistance in experimental models of pediatric acute lymphoblastic leukemia. Cancer Research. 2007;**67**(9):4482-4490

signaling cascades mediate distinct glucocorticoid resistance mechanisms

[74] Serafin V et al. Glucocorticoid resistance is reverted by LCK inhibition in pediatric T-cell acute lymphoblastic leukemia. Blood.

2005;**93**(2-5):153-160

2013;**24**(6):766-776

2010;**115**(23):4810-4819

[73] Jones CL et al. MAPK

2017;**130**(25):2750-2761

2012;**7**(11):e49926

[75] Nagao K, Iwai Y, Miyashita T. RCAN1 is an important mediator of glucocorticoid-induced apoptosis in human leukemic cells. PLoS One.

[76] Cialfi S et al. Glucocorticoid sensitivity of T-cell lymphoblastic leukemia/lymphoma is associated with glucocorticoid receptor-mediated inhibition of Notch1 expression. Leukemia. 2013;**27**(2):485-488

in pediatric leukemia. Blood. 2015;**126**(19):2202-2212

*DOI: http://dx.doi.org/10.5772/intechopen.84184*

binding across the human genome. Cell.

[61] Swinstead EE et al. Steroid receptors reprogram FoxA1 occupancy through dynamic chromatin transitions. Cell.

2016;**166**(5):1269-1281 e19

2017;**23**(4):424-428

2016;**165**(3):593-605

2017;**45**(4):1805-1819

2010;**120**(4-5):218-227

2016;**63**(8):1457-1460

2017;**17**(4):233-247

[62] Love MI et al. Role of the

[63] Gross KL, Lu NZ, Cidlowski JA. Molecular mechanisms regulating

[64] Wasim M et al. PLZF/ZBTB16, a glucocorticoid response gene in acute lymphoblastic leukemia, interferes with glucocorticoid-induced apoptosis. The Journal of Steroid Biochemistry and Molecular Biology.

[65] Klein K et al. Glucocorticoidinduced proliferation in untreated pediatric acute myeloid leukemic blasts. Pediatric Blood & Cancer.

[66] Gruver-Yates AL, Cidlowski JA. Tissue-specific actions of

glucocorticoids on apoptosis: A doubleedged sword. Cell. 2013;**2**(2):202-223

[67] Cain DW, Cidlowski JA. Immune regulation by glucocorticoids. Nature Reviews. Immunology.

[68] Ploner C et al. Glucocorticoidinduced apoptosis and glucocorticoid

glucocorticoid sensitivity and resistance. Molecular and Cellular Endocrinology. 2009;**300**(1-2):7-16

chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation. Nucleic Acids Research.

[60] Guo B et al. Glucocorticoid hormone-induced chromatin remodeling enhances human hematopoietic stem cell homing and engraftment. Nature Medicine. *Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.84184*

binding across the human genome. Cell. 2016;**166**(5):1269-1281 e19

*Germ Line Mutations Associated Leukemia*

RJ. Glucocorticoids, Milestones in drug therapy. Basel, Switzerland: Boston: Birkhäuser Verlag; 2001. p. 205

at most promoters in human cells. Cell.

[50] Li G et al. Extensive promotercentered chromatin interactions provide a topological basis for transcription regulation. Cell. 2012;**148**(1-2):84-98

[51] Corces MR et al. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nature Genetics.

[53] Thurman RE et al. The accessible chromatin landscape of the human genome. Nature. 2012;**489**(7414):75-82

[54] Perera D et al. Differential DNA repair underlies mutation hotspots at active promoters in cancer genomes. Nature. 2016;**532**(7598):259-263

[55] Greenstein S et al. Mechanisms of glucocorticoid-mediated apoptosis in hematological malignancies. Clinical Cancer Research. 2002;**8**(6):1681-1694

[56] Watson LC et al. The glucocorticoid receptor dimer interface allosterically transmits sequence-specific DNA signals. Nature Structural & Molecular

Biology. 2013;**20**(7):876-883

2008;**29**(5):611-624

2010;**24**(3):511-525

[57] John S et al. Interaction of the glucocorticoid receptor with the chromatin landscape. Molecular Cell.

[58] Paakinaho V et al. Glucocorticoid receptor activates poised FKBP51 locus through long-distance

interactions. Molecular Endocrinology.

[59] Vockley CM et al. Direct GR binding sites potentiate clusters of TF

2007;**130**(1):77-88

2016;**48**(10):1193-1203

2016;**167**(5):1188-1200

[52] Hnisz D, Day DS, Young RA. Insulated neighborhoods: Structural and functional units of mammalian gene control. Cell.

[40] Dalakas MC. Inflammatory muscle diseases. The New England Journal of Medicine. 2015;**372**(18):1734-1747

[41] Nair P et al. Oral glucocorticoidsparing effect of benralizumab in severe asthma. The New England Journal of Medicine. 2017;**376**(25):2448-2458

[42] Inaba H, Pui CH. Glucocorticoid

[43] Kim IK et al. Glucocorticoidinduced tumor necrosis factor

receptor-related protein co-stimulation facilitates tumor regression by inducing IL-9-producing helper T cells. Nature Medicine. 2015;**21**(9):1010-1017

[44] Palumbo A et al. Daratumumab, bortezomib, and dexamethasone for multiple myeloma. The New England Journal of Medicine.

[45] Pui CH, Evans WE. A 50-year journey to cure childhood acute lymphoblastic leukemia. Seminars in Hematology. 2013;**50**(3):185-196

[46] Klumper E et al. In vitro cellular drug resistance in children with

[47] Howard SC et al. Urolithiasis in pediatric patients with acute lymphoblastic leukemia. Leukemia.

[48] Australian Institute of Health and Welfare. A Picture of Australia's Children 2012. Canberra: Australian Institute of Health and Welfare; 2012

[49] Guenther MG et al. A chromatin landmark and transcription initiation

2003;**17**(3):541-546

relapsed/refractory acute lymphoblastic leukemia. Blood. 1995;**86**(10):3861-3868

use in acute lymphoblastic leukaemia. The Lancet Oncology.

2010;**11**(11):1096-1106

2016;**375**(8):754-766

[39] Goulding NJ, Flower

**28**

[60] Guo B et al. Glucocorticoid hormone-induced chromatin remodeling enhances human hematopoietic stem cell homing and engraftment. Nature Medicine. 2017;**23**(4):424-428

[61] Swinstead EE et al. Steroid receptors reprogram FoxA1 occupancy through dynamic chromatin transitions. Cell. 2016;**165**(3):593-605

[62] Love MI et al. Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation. Nucleic Acids Research. 2017;**45**(4):1805-1819

[63] Gross KL, Lu NZ, Cidlowski JA. Molecular mechanisms regulating glucocorticoid sensitivity and resistance. Molecular and Cellular Endocrinology. 2009;**300**(1-2):7-16

[64] Wasim M et al. PLZF/ZBTB16, a glucocorticoid response gene in acute lymphoblastic leukemia, interferes with glucocorticoid-induced apoptosis. The Journal of Steroid Biochemistry and Molecular Biology. 2010;**120**(4-5):218-227

[65] Klein K et al. Glucocorticoidinduced proliferation in untreated pediatric acute myeloid leukemic blasts. Pediatric Blood & Cancer. 2016;**63**(8):1457-1460

[66] Gruver-Yates AL, Cidlowski JA. Tissue-specific actions of glucocorticoids on apoptosis: A doubleedged sword. Cell. 2013;**2**(2):202-223

[67] Cain DW, Cidlowski JA. Immune regulation by glucocorticoids. Nature Reviews. Immunology. 2017;**17**(4):233-247

[68] Ploner C et al. Glucocorticoidinduced apoptosis and glucocorticoid resistance in acute lymphoblastic leukemia. The Journal of Steroid Biochemistry and Molecular Biology. 2005;**93**(2-5):153-160

[69] Schmidt S et al. Glucocorticoid resistance in two key models of acute lymphoblastic leukemia occurs at the level of the glucocorticoid receptor. The FASEB Journal. 2006;**20**(14):2600-2602

[70] Piovan E et al. Direct reversal of glucocorticoid resistance by AKT inhibition in acute lymphoblastic leukemia. Cancer Cell. 2013;**24**(6):766-776

[71] van Galen JC et al. BTG1 regulates glucocorticoid receptor autoinduction in acute lymphoblastic leukemia. Blood. 2010;**115**(23):4810-4819

[72] Bachmann PS et al. Divergent mechanisms of glucocorticoid resistance in experimental models of pediatric acute lymphoblastic leukemia. Cancer Research. 2007;**67**(9):4482-4490

[73] Jones CL et al. MAPK signaling cascades mediate distinct glucocorticoid resistance mechanisms in pediatric leukemia. Blood. 2015;**126**(19):2202-2212

[74] Serafin V et al. Glucocorticoid resistance is reverted by LCK inhibition in pediatric T-cell acute lymphoblastic leukemia. Blood. 2017;**130**(25):2750-2761

[75] Nagao K, Iwai Y, Miyashita T. RCAN1 is an important mediator of glucocorticoid-induced apoptosis in human leukemic cells. PLoS One. 2012;**7**(11):e49926

[76] Cialfi S et al. Glucocorticoid sensitivity of T-cell lymphoblastic leukemia/lymphoma is associated with glucocorticoid receptor-mediated inhibition of Notch1 expression. Leukemia. 2013;**27**(2):485-488

[77] Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nature Reviews. Drug Discovery. 2014;**13**(9):673-691

[78] Stresemann C, Lyko F. Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine. International Journal of Cancer. 2008;**123**(1):8-13

[79] Al-Romaih K et al. Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation. Cancer Cell International. 2007;**7**:14

[80] Benton CB et al. Safety and clinical activity of 5-aza-2′-deoxycytidine (decitabine) with or without hyper-CVAD in relapsed/refractory acute lymphocytic leukaemia. British Journal of Haematology. 2014;**167**(3):356-365

[81] Garcia-Manero G et al. DNA methylation of multiple promoterassociated CpG islands in adult acute lymphocytic leukemia. Clinical Cancer Research. 2002;**8**(7):2217-2224

[82] Garcia-Manero G et al. Epigenetics of acute lymphocytic leukemia. Seminars in Hematology. 2009;**46**(1):24-32. DOI: 10.1053/j. seminhematol.2008.09.008

[83] Heerboth S et al. Use of epigenetic drugs in disease: An overview. Genetics & Epigenetics. 2014;**6**:9-19

[84] Lu BY et al. Decitabine enhances chemosensitivity of early T-cell precursor-acute lymphoblastic leukemia cell lines and patient-derived samples. Leukemia & Lymphoma. 2016;**57**(8):1938-1941

[85] Moreno DA et al. Research paper: Differential expression of HDAC3,

HDAC7 and HDAC9 is associated with prognosis and survival in childhood acute lymphoblastic leukaemia. British Journal of Haematology. 2010;**150**(6):665-673

[86] Fujisawa T, Filippakopoulos P. Functions of bromodomaincontaining proteins and their roles in homeostasis and cancer. Nature Reviews Molecular Cell Biology. 2017;**18**:246

[87] Dey A et al. Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription. Molecular Biology of the Cell. 2009;**20**(23):4899-4909

[88] Choi SK et al. JQ1, an inhibitor of the epigenetic reader BRD4, suppresses the bidirectional MYC-AP4 axis via multiple mechanisms. Oncology Reports. 2016;**35**(2):1186

[89] Delmore JE et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell. 2011;**146**(6):904-917

[90] Jung P, Hermeking H. The c-MYC-AP4-p21 cascade. Cell Cycle. 2009;**8**(7):982-989

[91] Lock RB et al. Epigenetic silencing of the pro-apoptotic Bim gene in glucocorticoid poor-responsive pediatric acute lymphoblastic leukemia, and its reversal by histone deacetylase inhibition. Blood. 2009;**114**(22):939

[92] Zhang C et al. Histone acetylation: Novel target for the treatment of acute lymphoblastic leukemia. Clinical Epigenetics. 2015;**7**:117

[93] Leclerc GJ et al. Histone deacetylase inhibitors induce FPGS mRNA expression and intracellular accumulation of long-chain methotrexate polyglutamates in childhood acute lymphoblastic leukemia: Implications for combination therapy. Leukemia. 2010;**24**(3):552

**31**

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics*

*DOI: http://dx.doi.org/10.5772/intechopen.84184*

[94] Cancer Genome Atlas Research, N et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England Journal of Medicine. 2013;**368**(22):2059-2074

[95] Hnisz D et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science.

[96] Iacobucci I, Mullighan CG. Genetic basis of acute lymphoblastic leukemia.

[97] Australian Institute of Health and Welfare (AIHW). Australian Cancer Incidence and Mortality (ACIM) Books: Acute Lymphoblastic Leukaemia.

2016;**351**(6280):1454-1458

Journal of Clinical Oncology.

2017;**35**(9):975-983

Canberra: AIHW; 2014

*Epigenetic Landscape in Leukemia and Its Impact on Antileukemia Therapeutics DOI: http://dx.doi.org/10.5772/intechopen.84184*

[94] Cancer Genome Atlas Research, N et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England Journal of Medicine. 2013;**368**(22):2059-2074

*Germ Line Mutations Associated Leukemia*

HDAC7 and HDAC9 is associated with prognosis and survival in childhood acute lymphoblastic leukaemia. British Journal of Haematology.

[86] Fujisawa T, Filippakopoulos P. Functions of bromodomaincontaining proteins and their roles in homeostasis and cancer. Nature Reviews Molecular Cell Biology. 2017;**18**:246

[87] Dey A et al. Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription. Molecular Biology of the Cell. 2009;**20**(23):4899-4909

[88] Choi SK et al. JQ1, an inhibitor of the epigenetic reader BRD4, suppresses the bidirectional MYC-AP4 axis via multiple mechanisms. Oncology

[89] Delmore JE et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell. 2011;**146**(6):904-917

[91] Lock RB et al. Epigenetic silencing of the pro-apoptotic Bim gene in glucocorticoid poor-responsive

pediatric acute lymphoblastic leukemia, and its reversal by histone deacetylase inhibition. Blood. 2009;**114**(22):939

[92] Zhang C et al. Histone acetylation: Novel target for the treatment of acute lymphoblastic leukemia. Clinical

leukemia: Implications for combination therapy. Leukemia. 2010;**24**(3):552

Reports. 2016;**35**(2):1186

[90] Jung P, Hermeking H. The c-MYC-AP4-p21 cascade. Cell Cycle.

2009;**8**(7):982-989

Epigenetics. 2015;**7**:117

[93] Leclerc GJ et al. Histone deacetylase inhibitors induce FPGS mRNA expression and intracellular

accumulation of long-chain methotrexate polyglutamates in childhood acute lymphoblastic

2010;**150**(6):665-673

[78] Stresemann C, Lyko F. Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine. International Journal of Cancer.

[79] Al-Romaih K et al. Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation. Cancer Cell

[80] Benton CB et al. Safety and clinical activity of 5-aza-2′-deoxycytidine (decitabine) with or without hyper-CVAD in relapsed/refractory acute lymphocytic leukaemia. British Journal of Haematology. 2014;**167**(3):356-365

[81] Garcia-Manero G et al. DNA methylation of multiple promoterassociated CpG islands in adult acute lymphocytic leukemia. Clinical Cancer

Research. 2002;**8**(7):2217-2224

[83] Heerboth S et al. Use of epigenetic drugs in disease: An overview. Genetics

[84] Lu BY et al. Decitabine enhances chemosensitivity of early T-cell precursor-acute lymphoblastic

leukemia cell lines and patient-derived samples. Leukemia & Lymphoma.

[85] Moreno DA et al. Research paper: Differential expression of HDAC3,

[82] Garcia-Manero G et al. Epigenetics of acute lymphocytic leukemia. Seminars in Hematology. 2009;**46**(1):24-32. DOI: 10.1053/j. seminhematol.2008.09.008

& Epigenetics. 2014;**6**:9-19

2016;**57**(8):1938-1941

[77] Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nature Reviews. Drug Discovery.

2014;**13**(9):673-691

2008;**123**(1):8-13

International. 2007;**7**:14

**30**

[95] Hnisz D et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science. 2016;**351**(6280):1454-1458

[96] Iacobucci I, Mullighan CG. Genetic basis of acute lymphoblastic leukemia. Journal of Clinical Oncology. 2017;**35**(9):975-983

[97] Australian Institute of Health and Welfare (AIHW). Australian Cancer Incidence and Mortality (ACIM) Books: Acute Lymphoblastic Leukaemia. Canberra: AIHW; 2014

**33**

Section 3

Germ Line Mutations

Associated Leukemia

Section 3

## Germ Line Mutations Associated Leukemia

**35**

**Chapter 3**

**Abstract**

the affected families.

**1. Introduction**

Familial Leukemia Associated

Familial predisposition to leukemia has been known for decades. In some families, this condition is also associated with thrombocytopenia and history of bleeding. Germline mutations in the RUNX1 gene have been proven to cause familial platelet disorder with predisposition to myeloid malignancies (FDPMM). The disease typically presents with mild-to-moderate thrombocytopenia with normal-size platelets, functional platelet defects leading to prolonged bleeding, and an increased risk to develop myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), or T-cell acute lymphoblastic leukemia (T-ALL). In recent years, molecular defects in other genes, such as ANKRD26 and ETV6, have been associated with thrombocytopenia and susceptibility to hematological malignancy as well. In our chapter, we will present a review of up-to-date knowledge on this topic along with several case studies demonstrating the diagnostic process and management of

Familial leukemia (e.g., repeated occurrence of hematologic neoplasia in families more often than is expected by chance alone) has been a topic of interest for decades. Almost a hundred years ago, connections between inherited forms of myelodysplastic syndrome (MDS) and myeloid and lymphoid leukemia were established with several constitutional disorders in childhood, such as Fanconi anemia [1]. Since then, a number of additional inherited bone marrow failure syndromes and inherited conditions with predisposition to leukemia were discovered. Repeated occurrence of similar phenotypes, high clinical penetrance for hematologic disorders, and often consanguineous inheritance made identification of the respective genetic causes easier. These conditions are caused by germline mutations (genetic changes which can be carried on to next generations) in genes playing an important role in the development and maintenance of hematopoietic system. Collective effort of many researchers in the past has improved the knowledge about risk for MDS/leukemia, as well as the natural history and clinical outcomes of affected patients [2]. Some of these syndromes present with a distinctive hematological phenotype thrombocytopenia. Until the end of the last century, only a few forms of inherited thrombocytopenia were known, all of which were extremely rare. Since then, the knowledge of thrombocytopenia has improved, and we presently know at least 26 disorders caused by mutations in 30 genes [3]. It also became quite apparent that in

with Thrombocytopenia

*Jakub Trizuljak and Michael Doubek*

**Keywords:** AML, familial, RUNX1, ANKRD26, ETV6

#### **Chapter 3**

## Familial Leukemia Associated with Thrombocytopenia

*Jakub Trizuljak and Michael Doubek*

#### **Abstract**

Familial predisposition to leukemia has been known for decades. In some families, this condition is also associated with thrombocytopenia and history of bleeding. Germline mutations in the RUNX1 gene have been proven to cause familial platelet disorder with predisposition to myeloid malignancies (FDPMM). The disease typically presents with mild-to-moderate thrombocytopenia with normal-size platelets, functional platelet defects leading to prolonged bleeding, and an increased risk to develop myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), or T-cell acute lymphoblastic leukemia (T-ALL). In recent years, molecular defects in other genes, such as ANKRD26 and ETV6, have been associated with thrombocytopenia and susceptibility to hematological malignancy as well. In our chapter, we will present a review of up-to-date knowledge on this topic along with several case studies demonstrating the diagnostic process and management of the affected families.

**Keywords:** AML, familial, RUNX1, ANKRD26, ETV6

#### **1. Introduction**

Familial leukemia (e.g., repeated occurrence of hematologic neoplasia in families more often than is expected by chance alone) has been a topic of interest for decades. Almost a hundred years ago, connections between inherited forms of myelodysplastic syndrome (MDS) and myeloid and lymphoid leukemia were established with several constitutional disorders in childhood, such as Fanconi anemia [1]. Since then, a number of additional inherited bone marrow failure syndromes and inherited conditions with predisposition to leukemia were discovered. Repeated occurrence of similar phenotypes, high clinical penetrance for hematologic disorders, and often consanguineous inheritance made identification of the respective genetic causes easier. These conditions are caused by germline mutations (genetic changes which can be carried on to next generations) in genes playing an important role in the development and maintenance of hematopoietic system. Collective effort of many researchers in the past has improved the knowledge about risk for MDS/leukemia, as well as the natural history and clinical outcomes of affected patients [2].

Some of these syndromes present with a distinctive hematological phenotype thrombocytopenia. Until the end of the last century, only a few forms of inherited thrombocytopenia were known, all of which were extremely rare. Since then, the knowledge of thrombocytopenia has improved, and we presently know at least 26 disorders caused by mutations in 30 genes [3]. It also became quite apparent that in


#### **Figure 1.**

*Myeloid malignancies with germline predisposition. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia [4].*

some families, there is a connection between thrombocytopenia and additional risk of hematological malignancy. Thanks to availability of next-generation sequencing (NGS) technologies, genes associated with hereditary thrombocytopenia and risk of leukemic transformation were successfully identified, notably RUNX1, ETV6, and ANKRD26. These new hereditary syndromes were included in the 2016 revision of World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia [4] **(Figure 1)**.

#### **2.** *RUNX1* **deficiency (familial platelet disorder with predisposition to myeloid malignancies (FPDMM))**

RUNT-related transcription factor 1 (RUNX1) is a master regulator of hematopoiesis [5]. It is involved in the most frequent chromosome translocations in leukemia (i.e., t (12;21)/*RUNX1/ETV6*, t(8;21)/*RUNX1/RUNX1T1*, and t(3;21)/*RUNX1/ EVI1*) [6]. Moreover, somatic *RUNX1* mutations have been identified as recurrent abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) [7].

For the first time, germline *RUNX1* mutations were described in 1999 [8]. Individuals carrying germline *RUNX1* mutation may develop familial platelet disorder with predisposition to myeloid malignancies (FPDMM). Characteristic features include (1) thrombocytopenia, (2) functional platelet defects, and (3) an increased risk to develop MDS, AML, or acute T-lymphoblastic leukemia (T-ALL). There is a significant phenotypic heterogeneity. FPDMM (MIM601399) is inherited in an autosomal-dominant mode with incomplete penetrance and variable expressivity [5, 6].

**37**

*Familial Leukemia Associated with Thrombocytopenia DOI: http://dx.doi.org/10.5772/intechopen.85303*

**2.1 Diagnostic criteria to identify at-risk individuals**

to prevent adverse outcomes after transplantation [15–17].

marrow smears even before leukemic transformation [20].

**2.3 Role of** *RUNX1* **in hematopoiesis**

patients are likely candidates for FDPMM [18].

**2.2 Platelet features**

Diagnosis of FDPMM in patients with leukemia carries important clinical implications for the patient but also for her/his family. Recognition of clinical features pointing to this genetic predisposition is crucial. The most important feature is persistent thrombocytopenia or aspirin-like platelet disorder. Pedigree analysis can identify first- or second-degree relatives with higher occurrence of bleeding and hematological malignancies. The bleeding symptoms may be mild or not present. Onset of leukemia varies and spans from infant age to adulthood [9, 10]. In the case of family history of MDS, early-onset leukemia and/or a personal history of bleeding, immune deficiency, or dysmorphic features, genetic counseling is advised [11, 12]. Comprehensive evaluation involves a thorough review of individual's family and personal history, hematologic investigation, and personal risk assessment of likelihood of a hereditary predisposition within his/her family, and if necessary, genetic testing with NGS to determine the possibility of a germline mutation should be offered [13]. We provide an example of a familial case of FDPMM in **Figure 2** [14]. Predictive testing of healthy relatives is advised due to risk of bleeding and leukemia, even in infancy. In the case of individuals with leukemia, where allogenic stem cell transplantation from a HLA-matching sibling donor is the best possible treatment option, mutation screening should be a part of decision-making process,

Due to the advance and widespread use of NGS technologies in diagnosis of myeloid neoplasia in recent years, many individuals at risk are being identified by screening large cohorts of patients. In particular, leukemias with homozygous RUNX1 mutations, biallelic RUNX1 mutations, and trisomy 21 indicate that the

A personal or family history of thrombocytopenia and/or bleeding tendency may be an important pointer to diagnose FDPMM in patient with MDS, AML, or T-ALL. The platelet count is usually mild to moderate and, in some cases, low-normal and even normal. Platelet size is not affected—similar to ETV6- or ANKRD26-related thrombocytopenias, which are also characterized by normal-size platelets [19]. Thrombocytopenia is caused by abnormal megakaryocyte maturation and impaired proplatelet formation. Dysmegakaryopoiesis may be present in bone

A functional defect of platelets is present in most, if not all, patients with RUNX1 germline mutations, leading to abnormal secretion and aggregation [21]. The bleeding diathesis is variable within and among families. As some carriers of RUNX1 have mild or none bleeding symptoms, the presence of mutation may go unnoticed, and genetic screening is necessary to determine mutational status.

The finding of platelet abnormalities in patients with FDPMM has revealed the essential role of RUNX1 in the megakaryocytic lineage. RUNX1 works as a transcription factor at different stages of megakaryocyte development by regulating the expression of multiple factors relevant to platelet production and function. Reduced expression of RUNX1 target genes, including MPL proto-oncogene, thrombopoietin receptor (MPL), nonmuscle myosin IIA/myosin heavy chain 9 (MYH9) and its regulatory chain MLC2, arachidonate 12-lipoxygenase (ALOX12), and NFE2, has been shown to cause the defect in platelet number and function in FDPMM [22, 23]. What is more, increased levels

*Germ Line Mutations Associated Leukemia*

acute leukemia [4] **(Figure 1)**.

**myeloid malignancies (FPDMM))**

*classification of myeloid neoplasms and acute leukemia [4].*

some families, there is a connection between thrombocytopenia and additional risk of hematological malignancy. Thanks to availability of next-generation sequencing (NGS) technologies, genes associated with hereditary thrombocytopenia and risk of leukemic transformation were successfully identified, notably RUNX1, ETV6, and ANKRD26. These new hereditary syndromes were included in the 2016 revision of World Health Organization (WHO) classification of myeloid neoplasms and

*Myeloid malignancies with germline predisposition. The 2016 revision to the World Health Organization* 

**2.** *RUNX1* **deficiency (familial platelet disorder with predisposition to** 

For the first time, germline *RUNX1* mutations were described in 1999 [8]. Individuals carrying germline *RUNX1* mutation may develop familial platelet disorder with predisposition to myeloid malignancies (FPDMM). Characteristic features include (1) thrombocytopenia, (2) functional platelet defects, and (3) an increased risk to develop MDS, AML, or acute T-lymphoblastic leukemia (T-ALL). There is a significant phenotypic heterogeneity. FPDMM (MIM601399) is inherited in an autosomal-dominant mode with incomplete penetrance and variable

RUNT-related transcription factor 1 (RUNX1) is a master regulator of hematopoiesis [5]. It is involved in the most frequent chromosome translocations in leukemia (i.e., t (12;21)/*RUNX1/ETV6*, t(8;21)/*RUNX1/RUNX1T1*, and t(3;21)/*RUNX1/ EVI1*) [6]. Moreover, somatic *RUNX1* mutations have been identified as recurrent abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukemia

**36**

(AML) [7].

**Figure 1.**

expressivity [5, 6].

#### **2.1 Diagnostic criteria to identify at-risk individuals**

Diagnosis of FDPMM in patients with leukemia carries important clinical implications for the patient but also for her/his family. Recognition of clinical features pointing to this genetic predisposition is crucial. The most important feature is persistent thrombocytopenia or aspirin-like platelet disorder. Pedigree analysis can identify first- or second-degree relatives with higher occurrence of bleeding and hematological malignancies. The bleeding symptoms may be mild or not present. Onset of leukemia varies and spans from infant age to adulthood [9, 10]. In the case of family history of MDS, early-onset leukemia and/or a personal history of bleeding, immune deficiency, or dysmorphic features, genetic counseling is advised [11, 12]. Comprehensive evaluation involves a thorough review of individual's family and personal history, hematologic investigation, and personal risk assessment of likelihood of a hereditary predisposition within his/her family, and if necessary, genetic testing with NGS to determine the possibility of a germline mutation should be offered [13]. We provide an example of a familial case of FDPMM in **Figure 2** [14].

Predictive testing of healthy relatives is advised due to risk of bleeding and leukemia, even in infancy. In the case of individuals with leukemia, where allogenic stem cell transplantation from a HLA-matching sibling donor is the best possible treatment option, mutation screening should be a part of decision-making process, to prevent adverse outcomes after transplantation [15–17].

Due to the advance and widespread use of NGS technologies in diagnosis of myeloid neoplasia in recent years, many individuals at risk are being identified by screening large cohorts of patients. In particular, leukemias with homozygous RUNX1 mutations, biallelic RUNX1 mutations, and trisomy 21 indicate that the patients are likely candidates for FDPMM [18].

#### **2.2 Platelet features**

A personal or family history of thrombocytopenia and/or bleeding tendency may be an important pointer to diagnose FDPMM in patient with MDS, AML, or T-ALL. The platelet count is usually mild to moderate and, in some cases, low-normal and even normal. Platelet size is not affected—similar to ETV6- or ANKRD26-related thrombocytopenias, which are also characterized by normal-size platelets [19]. Thrombocytopenia is caused by abnormal megakaryocyte maturation and impaired proplatelet formation. Dysmegakaryopoiesis may be present in bone marrow smears even before leukemic transformation [20].

A functional defect of platelets is present in most, if not all, patients with RUNX1 germline mutations, leading to abnormal secretion and aggregation [21].

The bleeding diathesis is variable within and among families. As some carriers of RUNX1 have mild or none bleeding symptoms, the presence of mutation may go unnoticed, and genetic screening is necessary to determine mutational status.

#### **2.3 Role of** *RUNX1* **in hematopoiesis**

The finding of platelet abnormalities in patients with FDPMM has revealed the essential role of RUNX1 in the megakaryocytic lineage. RUNX1 works as a transcription factor at different stages of megakaryocyte development by regulating the expression of multiple factors relevant to platelet production and function. Reduced expression of RUNX1 target genes, including MPL proto-oncogene, thrombopoietin receptor (MPL), nonmuscle myosin IIA/myosin heavy chain 9 (MYH9) and its regulatory chain MLC2, arachidonate 12-lipoxygenase (ALOX12), and NFE2, has been shown to cause the defect in platelet number and function in FDPMM [22, 23]. What is more, increased levels

#### **Figure 2.**

*Pedigree of a family with thrombocytopenia and predisposition to myeloid malignancies. We identified a family with platelet disorder and predisposition to myeloid malignancies. Using exome sequencing of samples of eight family members, we identified a pathogenic frameshift variant c.866delG (p.Gly289Aspfs\*22) in exon 8 of RUNX1 gene, resulting in a premature stop codon. The mutation occurs within the transactivation domain of RUNX1. One of the affected individuals developed myelodysplastic syndrome, which progressed to acute myelogenous leukemia. Platelet count (PLT) reported in ×109/L, samples analyzed by exome sequencing marked with an asterisk (\*), age of death if known [14].*

of nonmuscle myosin IIB (MYH10), which is physiologically repressed by RUNX1, contribute to thrombocytopenia by blocking megakaryocyte polyploidization [24].

RUNX1 is a master regulator in hematopoietic differentiation. It plays a role in the first wave of hematopoiesis producing primitive erythroid cells and megakaryocytes. By enhanced expression of CEBPE, it negatively regulates myeloid progenitors and induces granulocytic differentiation. RUNX1 also regulates cell adhesion to the bone marrow niche [25]. After dimerizing with core-binding factor beta (CBFB), RUNX1 binds to promotor regions of several transcription factors like PU.1, regulating their expression. Binding to DNA and CBFB occurs in the highly conserved Runt homology domain (RHD) at the N-terminal region. Transactivation occurs at the C-terminal part of the molecule [26].

#### **2.4 Phenotype/genotype correlation**

Most RUNX1 mutations lie in the Runt homology domain region (RHD) [1]. Causative mutations are deleterious—most often frameshift, nonsense, or in/del mutations that result in premature protein truncation or nonsense-mediated decay of mRNA. Missense mutations may be present as well. In these cases, it may be hard to determine the pathogenicity of found variants. Here, segregation analyses and functional analyses are needed to confirm the effect of the variants for pathogenesis. Loss of function mutations in RHD, located in the N-terminal part of the protein, impairs normal RUNX1 function by hindering dimerization and DNA binding. These, as well as mutations in the 5′ regulatory region cause haploinsufficiency [27]. Missense mutations in RHD and nonsense and frameshift mutations in the C-terminal domain may lead to dominant-negative effects [10].

What is more, there are inherited structural rearrangements involving RUNX1: FDPMM can also be caused by small deletions involving a few base pairs or single exons of the gene and large deletions leading to loss of the complete coding regions.

**39**

*Familial Leukemia Associated with Thrombocytopenia DOI: http://dx.doi.org/10.5772/intechopen.85303*

nucleotide polymorphism (SNP) arrays [11, 12].

nia, while others suffer from myeloid neoplasms [29].

trigger the leukemic transformation.

occurs at a younger age [13, 24].

WT1, and SRSF2 [33, 34].

**2.6 Clinical management**

Deletions of large parts of the long arm of chromosome 21 cause a contiguous gene with various clinical signs, e.g., facial dysmorphism, mental retardation, thrombocytopenia, and increased risk of myeloid malignancies. These large deletions can be reliably detected by array comparative genomic hybridization (CGH)/single-

There seems to be a higher risk of leukemic transformation in the case of dominant-negative mutations of RUNx1 as compared to loss-of-function mutations. Both types of alterations lead to thrombocytopenia phenotype, but only dominantnegative mutations enhance the proliferation rate and clonogenic potential [28]. In the case of haploinsufficiency, biallelic or second-hit mutations are needed to

Unfortunately, there is no clear phenotype/genotype correlation. Within one family, members carrying the same mutation may present with different clinical signs and severity of symptoms. Some carriers develop only mild thrombocytope-

**2.5 Risk of malignancy and second-hit mutations in RUNX1 deficiency**

atic mutation carriers, preceding overt MDS/AML or FDPMM [30].

The risk of malignant transformation into MDS or AML is estimated to be 30–40% [16]. Patients carrying dominant-negative RUNX1 mutations have a higher risk of malignant transformation. The spectrum of malignancies involves AML of various French-American-British subtypes and MDS (refractory anemia with excess blasts, chronic myelomonocytic leukemia and hypoplastic MDS with myelofibrosis). In some cases T-cell ALL has also been described. In the case of MDS/AML, age of onset is at an average of 33 years with a wide age range, while in T-cell ALL, it usually

During the course of the disease, the second allele may be inactivated, as expected for tumor suppressor genes according to two-hit hypothesis. Nowadays, there are no definitive answers to what triggers the malignant transformation in RUNX1 germline mutation carriers. However, clonal hematopoiesis may be present even in asymptom-

Carriers of RUNX1 germline mutations need additional genetic events to develop hematological neoplasm. Often, biallelic alterations of RUNX1 are found, due to secondary RUNx1 mutations or acquired trisomy 21 resulting in the duplication of the mutated allele [31]. RUNx1 mutations are associated with MLL partial tandem duplications, FLT3-ITD, IDH1/2, RAS mutations, and ETV6 rearrangements. These often occur in therapy-related AML [32]. Recently, malignant transformation was reported to be mediated by recurrent somatic mutations in CD25C gene in up to a half of a Japanese patient cohort with RUNX1-related myeloid neoplasia. Next-generation sequencing allows detection of additional mutations in known AML drivers, such as ASXL1, TET2, IDH1, CEBPD, RB1, MLI2, FLT3-ITD,

Treatment of RUNX1-related AML or MDS follows standard protocols. If a disease-causing germline mutation is known in the family, it is important to prevent

In families with high-penetrance mutations, regular clinical examinations including differential blood count are advised. In case of suspicious clinical symptoms or cytopenias, bone marrow aspiration or biopsy with morphological, cytogenetic, and molecular genetic investigations should be discussed. Using new NGS

hematopoietic stem cell transplantation from a sibling or other relative.

technologies, it is possible to follow up clonal hematopoiesis [30].

*Germ Line Mutations Associated Leukemia*

of nonmuscle myosin IIB (MYH10), which is physiologically repressed by RUNX1, contribute to thrombocytopenia by blocking megakaryocyte polyploidization [24]. RUNX1 is a master regulator in hematopoietic differentiation. It plays a role in the first wave of hematopoiesis producing primitive erythroid cells and megakaryocytes. By enhanced expression of CEBPE, it negatively regulates myeloid progenitors and induces granulocytic differentiation. RUNX1 also regulates cell adhesion to the bone marrow niche [25]. After dimerizing with core-binding factor beta (CBFB), RUNX1 binds to promotor regions of several transcription factors like PU.1, regulating their expression. Binding to DNA and CBFB occurs in the highly conserved Runt homology domain (RHD) at the N-terminal region. Transactivation

*Pedigree of a family with thrombocytopenia and predisposition to myeloid malignancies. We identified a family with platelet disorder and predisposition to myeloid malignancies. Using exome sequencing of samples of eight family members, we identified a pathogenic frameshift variant c.866delG (p.Gly289Aspfs\*22) in exon 8 of RUNX1 gene, resulting in a premature stop codon. The mutation occurs within the transactivation domain of RUNX1. One of the affected individuals developed myelodysplastic syndrome, which progressed to acute myelogenous leukemia. Platelet count (PLT) reported in ×109/L, samples analyzed by exome sequencing* 

Most RUNX1 mutations lie in the Runt homology domain region (RHD) [1]. Causative mutations are deleterious—most often frameshift, nonsense, or in/del mutations that result in premature protein truncation or nonsense-mediated decay of mRNA. Missense mutations may be present as well. In these cases, it may be hard to determine the pathogenicity of found variants. Here, segregation analyses and functional analyses are needed to confirm the effect of the variants for pathogenesis. Loss of function mutations in RHD, located in the N-terminal part of the protein, impairs normal RUNX1 function by hindering dimerization and DNA binding. These, as well as mutations in the 5′ regulatory region cause haploinsufficiency [27]. Missense mutations in RHD and nonsense and frameshift mutations

What is more, there are inherited structural rearrangements involving RUNX1: FDPMM can also be caused by small deletions involving a few base pairs or single exons of the gene and large deletions leading to loss of the complete coding regions.

in the C-terminal domain may lead to dominant-negative effects [10].

occurs at the C-terminal part of the molecule [26].

**2.4 Phenotype/genotype correlation**

*marked with an asterisk (\*), age of death if known [14].*

**38**

**Figure 2.**

Deletions of large parts of the long arm of chromosome 21 cause a contiguous gene with various clinical signs, e.g., facial dysmorphism, mental retardation, thrombocytopenia, and increased risk of myeloid malignancies. These large deletions can be reliably detected by array comparative genomic hybridization (CGH)/singlenucleotide polymorphism (SNP) arrays [11, 12].

There seems to be a higher risk of leukemic transformation in the case of dominant-negative mutations of RUNx1 as compared to loss-of-function mutations. Both types of alterations lead to thrombocytopenia phenotype, but only dominantnegative mutations enhance the proliferation rate and clonogenic potential [28]. In the case of haploinsufficiency, biallelic or second-hit mutations are needed to trigger the leukemic transformation.

Unfortunately, there is no clear phenotype/genotype correlation. Within one family, members carrying the same mutation may present with different clinical signs and severity of symptoms. Some carriers develop only mild thrombocytopenia, while others suffer from myeloid neoplasms [29].

#### **2.5 Risk of malignancy and second-hit mutations in RUNX1 deficiency**

The risk of malignant transformation into MDS or AML is estimated to be 30–40% [16]. Patients carrying dominant-negative RUNX1 mutations have a higher risk of malignant transformation. The spectrum of malignancies involves AML of various French-American-British subtypes and MDS (refractory anemia with excess blasts, chronic myelomonocytic leukemia and hypoplastic MDS with myelofibrosis). In some cases T-cell ALL has also been described. In the case of MDS/AML, age of onset is at an average of 33 years with a wide age range, while in T-cell ALL, it usually occurs at a younger age [13, 24].

During the course of the disease, the second allele may be inactivated, as expected for tumor suppressor genes according to two-hit hypothesis. Nowadays, there are no definitive answers to what triggers the malignant transformation in RUNX1 germline mutation carriers. However, clonal hematopoiesis may be present even in asymptomatic mutation carriers, preceding overt MDS/AML or FDPMM [30].

Carriers of RUNX1 germline mutations need additional genetic events to develop hematological neoplasm. Often, biallelic alterations of RUNX1 are found, due to secondary RUNx1 mutations or acquired trisomy 21 resulting in the duplication of the mutated allele [31]. RUNx1 mutations are associated with MLL partial tandem duplications, FLT3-ITD, IDH1/2, RAS mutations, and ETV6 rearrangements. These often occur in therapy-related AML [32]. Recently, malignant transformation was reported to be mediated by recurrent somatic mutations in CD25C gene in up to a half of a Japanese patient cohort with RUNX1-related myeloid neoplasia. Next-generation sequencing allows detection of additional mutations in known AML drivers, such as ASXL1, TET2, IDH1, CEBPD, RB1, MLI2, FLT3-ITD, WT1, and SRSF2 [33, 34].

#### **2.6 Clinical management**

Treatment of RUNX1-related AML or MDS follows standard protocols. If a disease-causing germline mutation is known in the family, it is important to prevent hematopoietic stem cell transplantation from a sibling or other relative.

In families with high-penetrance mutations, regular clinical examinations including differential blood count are advised. In case of suspicious clinical symptoms or cytopenias, bone marrow aspiration or biopsy with morphological, cytogenetic, and molecular genetic investigations should be discussed. Using new NGS technologies, it is possible to follow up clonal hematopoiesis [30].

#### **2.7 Conclusion**

RUNX1 deficiency is a myeloid malignancy predisposition syndrome with high clinical penetrance and variable expressivity of its phenotypic effects. An aspirin-like platelet and mild-to-moderate thrombocytopenia are present in most of the patients. The presence of possible RUNX1 germline mutations should be part of decision-making process in management of HSCT and donor choice in MDS/ AML. Follow-up of asymptomatic mutation carriers is necessary.

#### **3. ETV6-related thrombocytopenia with propensity to hematological malignancies**

ETV6 was originally discovered in a leukemia-associated chromosomal translocation [35] and has subsequently been identified as a fusion partner in more than 30 chromosomal translocation oncogenes [36]. ETV6 is a transcriptional repressor that binds DNA via a C-terminal DNA-binding domain, highly conserved among ETS-family transcription factors [37]. The ETV6 N-terminal pointed (PNT) domain mediates self-association and frequently contributes to fusion proteins as the partner of tyrosine kinases [38]. Loss of ETV6 has firmly been implicated in the pathogenesis of ETV6-RUNX1(TEL-AML1)-associated childhood leukemia as there is invariably biallelic loss of ETV6 due to deletions of the second (nontranslocated) ETV6 allele [39].

More recently, genome-wide investigations have uncovered that ETV6 is subject to heterozygous mutations in hematologic malignancies, including myelodysplastic syndrome (MDS) [10, 11], acute myeloid leukemia (AML) [40], early T-cell precursor acute lymphoblastic leukemia (T-ALL) [41, 42], high-risk B-ALL [43], and diffuse large B-cell lymphoma (DLBCL) [44]. It remained unclear whether and how loss of ETV6 contributes to leukemogenesis.

Now a number of recent studies have expanded our knowledge. The initial report from Zhang et al. identified the link between heterozygous germline ETV6 mutation to dominantly inherited thrombocytopenia and predisposition to hematological malignancies [45]. Subsequent studies extended these findings to additional families with unique ETV6 germline mutations and predisposition to malignancy [46, 47]. With one exception, all of the germline mutations cluster within the highly conserved ETS domain. The only mutation outside the ETS domain, P214L, was repeatedly identified in family studies.

#### **3.1 Diagnostic criteria to identify at-risk individuals**

Diagnosis of ETV6-related thrombocytopenia is paramount due to clinical implications for the patient. The most important clinical feature is thrombocytopenia with normal-sized platelets. Sometimes, large mean corpuscular volume (MCV) of red blood cells is reported. In family history, individuals with occurrence of bleeding and hematological malignancies are identified. Bleeding symptoms are variable. No recurrent extra-hematologic abnormalities have been identified, though in some families, solid tumors may occur [45].

Genetic counseling, comprehensive evaluation of individual's family and personal history, hematologic investigation, personal risk assessment of likelihood of a hereditary predisposition within his/her family, and, if necessary, genetic testing with NGS are advised. In the case of a found mutation, predictive testing of healthy relatives is necessary to identify at-risk individuals [13]. We provide an example of a familial case of ETV6 deficiency in **Figure 3** [47]. In cases when allogenic

**41**

**Figure 3.**

*Familial Leukemia Associated with Thrombocytopenia DOI: http://dx.doi.org/10.5772/intechopen.85303*

**3.2 Platelet features**

of myeloid cells [48].

**3.3 Risk of malignancy**

ing between 100 and 150 × 109

relapse and transplant-related morbidity and mortality.

hematopoietic stem-cell transplantation is considered in a patient with leukemia and ETV6 mutation, possible sibling donors must be tested to avoid the risk of

All affected pedigrees with ETV6 germline mutations have a highly penetrant autosomal-dominant pattern of thrombocytopenia. Severity of thrombocytopenia is highly variable. Many patients have mild thrombocytopenia with platelet counts rang-

thrombocytopenia ˂20 × 109/L is seen rarely in the absence of myelodysplastic syndrome [46]. Bleeding symptoms reported are generally mild including petechiae, ecchymoses, epistaxis, gum bleeding, easy bruising, and menorrhagia. Platelet size is generally normal, though macrothrombocytopenia may be seen in a subset of patients. Hemoglobin is normal in most patients. Erythrocyte mean corpuscular volume (MCV) is generally normal or increased. Neutrophil counts are normal. Examination of bone marrow reveals frequent immature hypolobulated megakaryocytes, mild dyserythropoiesis, and mild hypolobulation and hypogranulation

A substantial number of patients carrying ETV6 germline mutation develop hematological malignancies during their lifetime. The risk of leukemic transformation is estimated to be up to 25–40%; the age of onset is highly variable (8–82 years). The spectrum of malignancies involves acute lymphoblastic leukemia (ALL) and myeloid malignancies including MDS, AML, chronic myelomonocytic leukemia (CMML), myeloproliferative disorders (typically polycythemia vera), and multiple myeloma. Special attention was brought to relationship between germline ETV6 mutations and childhood ALL. Targeted sequencing of

*Pedigree of a family with thrombocytopenia and occurrence of lymphoid and myeloid malignancies. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV), occurrence of B cell-precursor acute lymphoblastic leukemia (ALL) and myeloproliferative neoplasm (MPN). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ETS variant 6), c.1138T>A, encoding a p.Trp380Arg substitution in the C-terminal DNA-binding domain, segregating with thrombocytopenia and elevated MCV. The role of Trp380 is structural, being surrounded by hydrophobic residues in the domain hydrophobic core. Its substitution by an arginine will therefore severely destabilize the domain structure. Platelet count (PLT) reported in ×109/L, samples analyzed by exome sequencing marked with an asterisk [47].*

/L, while others had platelet counts ˂50 × 109

/L. Severe

hematopoietic stem-cell transplantation is considered in a patient with leukemia and ETV6 mutation, possible sibling donors must be tested to avoid the risk of relapse and transplant-related morbidity and mortality.

#### **3.2 Platelet features**

*Germ Line Mutations Associated Leukemia*

RUNX1 deficiency is a myeloid malignancy predisposition syndrome with high clinical penetrance and variable expressivity of its phenotypic effects. An aspirin-like platelet and mild-to-moderate thrombocytopenia are present in most of the patients. The presence of possible RUNX1 germline mutations should be part of decision-making process in management of HSCT and donor choice in MDS/

**3. ETV6-related thrombocytopenia with propensity to hematological** 

ETV6 was originally discovered in a leukemia-associated chromosomal translocation [35] and has subsequently been identified as a fusion partner in more than 30 chromosomal translocation oncogenes [36]. ETV6 is a transcriptional repressor that binds DNA via a C-terminal DNA-binding domain, highly conserved among ETS-family transcription factors [37]. The ETV6 N-terminal pointed (PNT) domain mediates self-association and frequently contributes to fusion proteins as the partner of tyrosine kinases [38]. Loss of ETV6 has firmly been implicated in the pathogenesis of ETV6-RUNX1(TEL-AML1)-associated childhood leukemia as there is invariably biallelic loss of ETV6 due to deletions of the second (nontranslocated)

More recently, genome-wide investigations have uncovered that ETV6 is subject to heterozygous mutations in hematologic malignancies, including myelodysplastic syndrome (MDS) [10, 11], acute myeloid leukemia (AML) [40], early T-cell precursor acute lymphoblastic leukemia (T-ALL) [41, 42], high-risk B-ALL [43], and diffuse large B-cell lymphoma (DLBCL) [44]. It remained unclear whether and how

Now a number of recent studies have expanded our knowledge. The initial report from Zhang et al. identified the link between heterozygous germline ETV6 mutation to dominantly inherited thrombocytopenia and predisposition to hematological malignancies [45]. Subsequent studies extended these findings to additional families with unique ETV6 germline mutations and predisposition to malignancy [46, 47]. With one exception, all of the germline mutations cluster within the highly conserved ETS domain. The only mutation outside the ETS domain, P214L, was

Diagnosis of ETV6-related thrombocytopenia is paramount due to clinical implications for the patient. The most important clinical feature is thrombocytopenia with normal-sized platelets. Sometimes, large mean corpuscular volume (MCV) of red blood cells is reported. In family history, individuals with occurrence of bleeding and hematological malignancies are identified. Bleeding symptoms are variable. No recurrent extra-hematologic abnormalities have been identified, though in some

Genetic counseling, comprehensive evaluation of individual's family and personal history, hematologic investigation, personal risk assessment of likelihood of a hereditary predisposition within his/her family, and, if necessary, genetic testing with NGS are advised. In the case of a found mutation, predictive testing of healthy relatives is necessary to identify at-risk individuals [13]. We provide an example of a familial case of ETV6 deficiency in **Figure 3** [47]. In cases when allogenic

AML. Follow-up of asymptomatic mutation carriers is necessary.

**2.7 Conclusion**

**malignancies**

ETV6 allele [39].

loss of ETV6 contributes to leukemogenesis.

repeatedly identified in family studies.

families, solid tumors may occur [45].

**3.1 Diagnostic criteria to identify at-risk individuals**

**40**

All affected pedigrees with ETV6 germline mutations have a highly penetrant autosomal-dominant pattern of thrombocytopenia. Severity of thrombocytopenia is highly variable. Many patients have mild thrombocytopenia with platelet counts ranging between 100 and 150 × 109 /L, while others had platelet counts ˂50 × 109 /L. Severe thrombocytopenia ˂20 × 109/L is seen rarely in the absence of myelodysplastic syndrome [46]. Bleeding symptoms reported are generally mild including petechiae, ecchymoses, epistaxis, gum bleeding, easy bruising, and menorrhagia. Platelet size is generally normal, though macrothrombocytopenia may be seen in a subset of patients.

Hemoglobin is normal in most patients. Erythrocyte mean corpuscular volume (MCV) is generally normal or increased. Neutrophil counts are normal. Examination of bone marrow reveals frequent immature hypolobulated megakaryocytes, mild dyserythropoiesis, and mild hypolobulation and hypogranulation of myeloid cells [48].

#### **3.3 Risk of malignancy**

A substantial number of patients carrying ETV6 germline mutation develop hematological malignancies during their lifetime. The risk of leukemic transformation is estimated to be up to 25–40%; the age of onset is highly variable (8–82 years). The spectrum of malignancies involves acute lymphoblastic leukemia (ALL) and myeloid malignancies including MDS, AML, chronic myelomonocytic leukemia (CMML), myeloproliferative disorders (typically polycythemia vera), and multiple myeloma. Special attention was brought to relationship between germline ETV6 mutations and childhood ALL. Targeted sequencing of

#### **Figure 3.**

*Pedigree of a family with thrombocytopenia and occurrence of lymphoid and myeloid malignancies. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV), occurrence of B cell-precursor acute lymphoblastic leukemia (ALL) and myeloproliferative neoplasm (MPN). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ETS variant 6), c.1138T>A, encoding a p.Trp380Arg substitution in the C-terminal DNA-binding domain, segregating with thrombocytopenia and elevated MCV. The role of Trp380 is structural, being surrounded by hydrophobic residues in the domain hydrophobic core. Its substitution by an arginine will therefore severely destabilize the domain structure. Platelet count (PLT) reported in ×109/L, samples analyzed by exome sequencing marked with an asterisk [47].*

a large cohort of childhood ALL patients revealed 31 leukemia-associated ETV6 exonic variants [49]. All variants in this study were absent in control population. About 48% of found variants were found in the ETS DNA-binding domain and were predicted to be deleterious. Children with ETV6 variants were older at diagnosis (median 10.2 years) than those without ETV6 variants (4.7 years). There was no association between ETV6 mutation status and early treatment response or risk of relapse.

In some families, a few sold tumors have been reported: colorectal carcinoma, breast cancer, renal cell carcinoma, and tumor of the central nervous system. Further investigation is needed to understand the role of ETV6 in solid tumors [45, 49, 50].

#### **3.4 Mutation spectrum**

The mutation types in ETV6-related thrombocytopenia with predisposition to malignancies include nonsense, missense, splice site, and frameshift variants. The majority of mutations cluster within the ETS DNA-binding domain and are predicted to be deleterious. The p214L mutation, which resides in the linker region, has been recurrently identified in different families [45, 49].

#### **3.5 Second-hit mutations in ETV6 deficiency**

The development of leukemia with variable latency and incomplete penetrance suggests a need for further somatic mutations. Studies did not reveal mutations in the remaining wild-type ETV6 allele in most cases. Such examples are more of an exception. Acquisition of somatic defects in other genes, such as RUNX1, BCOR, and KRAS, is more prominent. The role of additional mutations in malignant transformation remains to be determined [45].

#### **3.6 Molecular structure and role of ETV6**

ETV6 is a part of a 26-member family of transcriptional regulators, defined by a highly conserved 85-amino-acid residue that mediates binding of target DNA. Different ETS factors can replace each other in the context of overexpression in vitro but exhibit functional diversity and individual specificity in DNA binding beyond the core motif. ETV6 has the capacity to form polymers with head-to-tail binding of two different protein surfaces within its PNT domain [51].

The primary function of ETV6 is a transcriptional repressor. The PNT domainmediated multimerization is required for high affinity DNA binding. Truncated ETV6 proteins resulting from frameshift mutations retaining either the PNT domain or the ETS domain were shown to exhibit a dominant-negative activity. This was also demonstrated for the familial germline mutations. This may suggest that the pathogenic activity of ETV6 mutations not only includes loss of function but also interferes with the wild-type allele [40, 52, 53].

ETV6 also plays an important role in embryonic development. Homozygous ETV6 germline disruption results in embryonic lethality in mice studies [54]. ETV6 is required for survival of hematopoietic stem cells in the bone marrow. It also promotes the late phases of megakaryopoiesis. Heterozygous disruption of ETV6 in mice is not associated with obvious phenotypes, implying the dominant-negative effect of germline mutations found in affected families: complete loss of ETV6 is lethal, but development of abnormalities requires more than heterozygous loss [55, 56].

**43**

*Familial Leukemia Associated with Thrombocytopenia DOI: http://dx.doi.org/10.5772/intechopen.85303*

**4. ANKRD26-related thrombocytopenia**

of hematologic malignancies was higher than expected.

**4.1 Diagnostic criteria, platelet features**

**4.2 Risk of malignancy**

Treatment of ETV6-related leukemia does not differ from standard protocols. As in FDP-MM, if a disease-causing germline mutation is known in the family, it is necessary to test siblings, as HSCT from a sibling carrier of ETV6 pathogenic variant should be avoided. Family members should be tested, and regular follow-up of mutation carriers including differential blood count is advised. Bone marrow aspiration and/or biopsy with thorough cytogenetic/molecular genetic investigation may be necessary in case of additional cytopenias or other suspicious clinical

Discovery of familial ETV6 germline mutations has established its clinical significance as a cause of thrombocytopenia, as well as a major cancer predisposition gene, associated with a substantial number of childhood B-ALL cases as well as myeloid malignancies. However, our understanding of the clinical impact of ETV6 mutations and physiological role of ETV6 remains incomplete. More work is needed to understand the molecular pathology of the mutations and stratify the risk of affected individuals.

Thrombocytopenia 2 (THC2 MIM 188000) is one of the rarest forms of autosomal-dominant thrombocytopenia. It has so far been reported only in 21 families across the world [57]. The THC2 locus was mapped to chromosome 10p11.1-p12 through linkage analysis in two independent studies [58, 59]. In the original studies, two missense changes in different linked genes were found to be causative of the disease: c.501G > C (p.Glu167Asp) and c.22 C > T (p.his8Tyr). Another study identified pedigrees with six additional ANKRD26 mutations, segregating with thrombocytopenia. All of them were located in a stretch of 19 nucleotides of the 5′ UTR that is highly conserved in evolution. These findings associate ANKRD26 5 ′ UTR mutations with thrombocytopenia [60]. Further reports extended the number of known families to 21 [61]. The abovementioned studies also found that the number

THC2-affected individuals have a degree of thrombocytopenia ranging from mild to severe and suffer from a mild bleeding diathesis. Major bleeding events are rare. Platelets are normal-sized and morphology does not reveal any defects. Examination of bone marrow shows dysmegakaryocytopoietic phenomena. No other changes in blood count, e.g., anemia and neutropenia, were reported [60].

A comprehensive study of 118 subjects affected with THC2 identified 10 patients

who developed myeloid malignancies: four acute myeloid leukemias (AML), four myelodysplastic syndromes, and two chronic myeloid leukemias (CML). Cumulative incidence of hematological malignancies in this subset of patients is 8.47%. The incidence of lymphoproliferative disorders and nonhematologic cancers was not higher than expected. Available data are compatible with the hypothesis

**3.7 Clinical management**

symptoms [13].

**3.8 Conclusion**

#### **3.7 Clinical management**

*Germ Line Mutations Associated Leukemia*

or risk of relapse.

**3.4 Mutation spectrum**

a large cohort of childhood ALL patients revealed 31 leukemia-associated ETV6 exonic variants [49]. All variants in this study were absent in control population. About 48% of found variants were found in the ETS DNA-binding domain and were predicted to be deleterious. Children with ETV6 variants were older at diagnosis (median 10.2 years) than those without ETV6 variants (4.7 years). There was no association between ETV6 mutation status and early treatment response

In some families, a few sold tumors have been reported: colorectal carcinoma, breast cancer, renal cell carcinoma, and tumor of the central nervous system. Further investigation is needed to understand the role of ETV6 in solid tumors [45, 49, 50].

The mutation types in ETV6-related thrombocytopenia with predisposition to malignancies include nonsense, missense, splice site, and frameshift variants. The majority of mutations cluster within the ETS DNA-binding domain and are predicted to be deleterious. The p214L mutation, which resides in the linker region, has

The development of leukemia with variable latency and incomplete penetrance suggests a need for further somatic mutations. Studies did not reveal mutations in the remaining wild-type ETV6 allele in most cases. Such examples are more of an exception. Acquisition of somatic defects in other genes, such as RUNX1, BCOR, and KRAS, is more prominent. The role of additional mutations in malignant

ETV6 is a part of a 26-member family of transcriptional regulators, defined by a highly conserved 85-amino-acid residue that mediates binding of target

specificity in DNA binding beyond the core motif. ETV6 has the capacity to form polymers with head-to-tail binding of two different protein surfaces within its PNT

The primary function of ETV6 is a transcriptional repressor. The PNT domainmediated multimerization is required for high affinity DNA binding. Truncated ETV6 proteins resulting from frameshift mutations retaining either the PNT

domain or the ETS domain were shown to exhibit a dominant-negative activity. This was also demonstrated for the familial germline mutations. This may suggest that the pathogenic activity of ETV6 mutations not only includes loss of function but

DNA. Different ETS factors can replace each other in the context of overexpression in vitro but exhibit functional diversity and individual

ETV6 also plays an important role in embryonic development.

Homozygous ETV6 germline disruption results in embryonic lethality in mice studies [54]. ETV6 is required for survival of hematopoietic stem cells in the bone marrow. It also promotes the late phases of megakaryopoiesis. Heterozygous disruption of ETV6 in mice is not associated with obvious phenotypes, implying the dominant-negative effect of germline mutations found in affected families: complete loss of ETV6 is lethal, but development of abnormalities requires more

been recurrently identified in different families [45, 49].

**3.5 Second-hit mutations in ETV6 deficiency**

transformation remains to be determined [45].

also interferes with the wild-type allele [40, 52, 53].

than heterozygous loss [55, 56].

**3.6 Molecular structure and role of ETV6**

**42**

domain [51].

Treatment of ETV6-related leukemia does not differ from standard protocols. As in FDP-MM, if a disease-causing germline mutation is known in the family, it is necessary to test siblings, as HSCT from a sibling carrier of ETV6 pathogenic variant should be avoided. Family members should be tested, and regular follow-up of mutation carriers including differential blood count is advised. Bone marrow aspiration and/or biopsy with thorough cytogenetic/molecular genetic investigation may be necessary in case of additional cytopenias or other suspicious clinical symptoms [13].

#### **3.8 Conclusion**

Discovery of familial ETV6 germline mutations has established its clinical significance as a cause of thrombocytopenia, as well as a major cancer predisposition gene, associated with a substantial number of childhood B-ALL cases as well as myeloid malignancies. However, our understanding of the clinical impact of ETV6 mutations and physiological role of ETV6 remains incomplete. More work is needed to understand the molecular pathology of the mutations and stratify the risk of affected individuals.

### **4. ANKRD26-related thrombocytopenia**

Thrombocytopenia 2 (THC2 MIM 188000) is one of the rarest forms of autosomal-dominant thrombocytopenia. It has so far been reported only in 21 families across the world [57]. The THC2 locus was mapped to chromosome 10p11.1-p12 through linkage analysis in two independent studies [58, 59]. In the original studies, two missense changes in different linked genes were found to be causative of the disease: c.501G > C (p.Glu167Asp) and c.22 C > T (p.his8Tyr). Another study identified pedigrees with six additional ANKRD26 mutations, segregating with thrombocytopenia. All of them were located in a stretch of 19 nucleotides of the 5′ UTR that is highly conserved in evolution. These findings associate ANKRD26 5 ′ UTR mutations with thrombocytopenia [60]. Further reports extended the number of known families to 21 [61]. The abovementioned studies also found that the number of hematologic malignancies was higher than expected.

#### **4.1 Diagnostic criteria, platelet features**

THC2-affected individuals have a degree of thrombocytopenia ranging from mild to severe and suffer from a mild bleeding diathesis. Major bleeding events are rare. Platelets are normal-sized and morphology does not reveal any defects. Examination of bone marrow shows dysmegakaryocytopoietic phenomena. No other changes in blood count, e.g., anemia and neutropenia, were reported [60].

#### **4.2 Risk of malignancy**

A comprehensive study of 118 subjects affected with THC2 identified 10 patients who developed myeloid malignancies: four acute myeloid leukemias (AML), four myelodysplastic syndromes, and two chronic myeloid leukemias (CML). Cumulative incidence of hematological malignancies in this subset of patients is 8.47%. The incidence of lymphoproliferative disorders and nonhematologic cancers was not higher than expected. Available data are compatible with the hypothesis

that ANKRD26-related thrombocytopenia predisposes to myeloid malignancy. However, penetrance for neoplasia is incomplete, and other genetic or environmental factors must contribute to development of these disorders [57].

#### **4.3 Molecular genetics**

ANKRD26 is the ancestor of a family of primate-specific genes termed POTE (prostate-ovary-testes- and placenta-expressed genes) whose expression is restricted to several normal to a few normal tissues and a larger number of malignancies, such as breast cancer. ANKRD26 is expressed also in megakaryocytes and to lesser extent erythroid cells [62, 63].

The functional role of ANKRD26 is unknown. Deleterious mutations of aNKRD26 in animal studies do not cause thrombocytopenia. This evidence suggests that THC2 is more likely to be caused by gain-of-function mutations rather than haploinsufficiency. It is suspected that mutations in the 5′ UTR interfere with mechanisms controlling the expression of ANKRD26 and affect megakaryopoiesis and platelet production, possibly by induction of apoptosis [60, 64].

#### **4.4 Clinical management**

As in the abovementioned entities, screening for ANKRD26 mutations must be a part of diagnostic process in hereditary thrombocytopenia and familial myeloid leukemia. Follow-up of asymptomatic mutation carriers in regular intervals including peripheral blood count and smear is necessary. The presence of ANKRD26 germline mutations in acute leukemia may also play a part in HSCT-related questions.

#### **4.5 Conclusions**

ANKRD26 is a rare form of inherited thrombocytopenia with low risk of bleeding and predisposition to myeloid malignancies. Recognition of this disorder is important in differential diagnosis of hereditary thrombocytopenia and proper management of affected subjects.

#### **5. Further candidate genes**

There are several genes associated with inherited bone marrow failure syndromes (IBMFS) and thrombocytopenia, notably MPL, THPO, HOXA11, MECOM, and RBM8A, as well as mutations in genes for X-linked thrombocytopenia and immune deficiency (GATA1, WAS) [65]. These clinical entities present with thrombopenia or pancytopenia and, in some cases, dysmorphic features. The IBMFS are complex disorders unified by development of bone marrow failure and increased risk of leukemic transformation. In some IBMFS, the steps toward leukemic transformation are better understood. In others, there is still much to learn. The estimated risk of malignancy in the abovementioned entities requires additional research.

MYH9 mutations result in congenital macrothrombocytopenia and predispose to kidney failure, hearing loss, and cataracts. There are a few published cases of germline mutations of MYH9 with myeloid malignancy [66]. Somatic expression of MYH9 has impact on overall survival in patients with AML [67]. However, additional studies on larger patient populations are needed to confirm this suspicion.

**45**

**Author details**

Brno, Czech Republic

provided the original work is properly cited.

Jakub Trizuljak1,2\* and Michael Doubek1,2

Hospital and Masaryk University, Brno, Czech Republic

\*Address all correspondence to: trizuljak.jakub@fnbrno.cz

*Familial Leukemia Associated with Thrombocytopenia DOI: http://dx.doi.org/10.5772/intechopen.85303*

Oncology, Central European Institute of Technology.

Masaryk University (grant MUNI/A/1105/2018).

The authors declare no conflict of interest.

In this chapter, we have summarized current knowledge of familial syndromes with thrombocytopenia and predisposition to hematologic malignancies. These rare disorders must be a part of differential diagnosis of (1) unexplained or familial thrombocytopenia, (2) myeloid malignancies with familial occurrence, and (3) bone marrow failure syndromes. Only a correct diagnosis with up-to-date hematological and molecular diagnostics can lead to proper follow-up of affected individuals and families. Personalized risk assessment must be made; and in the case of a familial germline mutation, genetic reproductive consultation should be offered.

This text was created with the help of my supervisor, Prof. Michael Doubek, MD, and colleagues from Department of Internal Medicine, Hematology and

This text was produced with the help of "Pomoc lidem s leukémií" foundation. This work was supported by Czech Ministry of Health (grant AZV 16-29447A) and

**6. Conclusion**

**Acknowledgements**

**Conflict of interest**

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Central European Institute of Technology (CEITEC), Masaryk University,

2 Department of Internal Medicine—Hematology and Oncology, University

### **6. Conclusion**

*Germ Line Mutations Associated Leukemia*

to lesser extent erythroid cells [62, 63].

**4.3 Molecular genetics**

**4.4 Clinical management**

questions.

**4.5 Conclusions**

management of affected subjects.

**5. Further candidate genes**

that ANKRD26-related thrombocytopenia predisposes to myeloid malignancy. However, penetrance for neoplasia is incomplete, and other genetic or environmen-

ANKRD26 is the ancestor of a family of primate-specific genes termed POTE

(prostate-ovary-testes- and placenta-expressed genes) whose expression is restricted to several normal to a few normal tissues and a larger number of malignancies, such as breast cancer. ANKRD26 is expressed also in megakaryocytes and

The functional role of ANKRD26 is unknown. Deleterious mutations of aNKRD26 in animal studies do not cause thrombocytopenia. This evidence suggests that THC2 is more likely to be caused by gain-of-function mutations rather than haploinsufficiency. It is suspected that mutations in the 5′ UTR interfere with mechanisms controlling the expression of ANKRD26 and affect megakaryopoiesis

As in the abovementioned entities, screening for ANKRD26 mutations must be a part of diagnostic process in hereditary thrombocytopenia and familial myeloid leukemia. Follow-up of asymptomatic mutation carriers in regular intervals including peripheral blood count and smear is necessary. The presence of ANKRD26 germline mutations in acute leukemia may also play a part in HSCT-related

ANKRD26 is a rare form of inherited thrombocytopenia with low risk of bleeding and predisposition to myeloid malignancies. Recognition of this disorder is important in differential diagnosis of hereditary thrombocytopenia and proper

There are several genes associated with inherited bone marrow failure syndromes (IBMFS) and thrombocytopenia, notably MPL, THPO, HOXA11, MECOM, and RBM8A, as well as mutations in genes for X-linked thrombocytopenia and immune deficiency (GATA1, WAS) [65]. These clinical entities present with thrombopenia or pancytopenia and, in some cases, dysmorphic features. The IBMFS are complex disorders unified by development of bone marrow failure and increased risk of leukemic transformation. In some IBMFS, the steps toward leukemic transformation are better understood. In others, there is still much to learn. The estimated risk of malignancy in the abovementioned entities requires additional

MYH9 mutations result in congenital macrothrombocytopenia and predispose to kidney failure, hearing loss, and cataracts. There are a few published cases of germline mutations of MYH9 with myeloid malignancy [66]. Somatic expression of MYH9 has impact on overall survival in patients with AML [67]. However, additional studies on larger patient populations are needed to confirm this

and platelet production, possibly by induction of apoptosis [60, 64].

tal factors must contribute to development of these disorders [57].

**44**

research.

suspicion.

In this chapter, we have summarized current knowledge of familial syndromes with thrombocytopenia and predisposition to hematologic malignancies. These rare disorders must be a part of differential diagnosis of (1) unexplained or familial thrombocytopenia, (2) myeloid malignancies with familial occurrence, and (3) bone marrow failure syndromes. Only a correct diagnosis with up-to-date hematological and molecular diagnostics can lead to proper follow-up of affected individuals and families. Personalized risk assessment must be made; and in the case of a familial germline mutation, genetic reproductive consultation should be offered.

### **Acknowledgements**

This text was created with the help of my supervisor, Prof. Michael Doubek, MD, and colleagues from Department of Internal Medicine, Hematology and Oncology, Central European Institute of Technology.

This text was produced with the help of "Pomoc lidem s leukémií" foundation. This work was supported by Czech Ministry of Health (grant AZV 16-29447A) and Masaryk University (grant MUNI/A/1105/2018).

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Jakub Trizuljak1,2\* and Michael Doubek1,2

1 Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic

2 Department of Internal Medicine—Hematology and Oncology, University Hospital and Masaryk University, Brno, Czech Republic

\*Address all correspondence to: trizuljak.jakub@fnbrno.cz

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Fanconi G. Familiäre infantile perniziosaartige Anämie (perniziöses Blutbild und Konstitution). Jahrbuch Kinderheilk. 1927;**117**:257-280

[2] Wlodarski MW, Niemeyer CM. Introduction: Genetic syndromes predisposing to myeloid neoplasia. Seminars in Hematology. 2017;**54**(2):57-59. DOI: 10.1053/j. seminhematol.2017.05.001

[3] Pecci A. Diagnosis and treatment of inherited thrombocytopenias. Clinical Genetics. 2016;**89**(2):141-153

[4] Arber DA, Orazi A, Hasserijan R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;**127**(20):2391-2405. DOI: 10.1182/blood-2016-03-643544

[5] Schlegelberger B, Heller PG. *RUNX1* deficiency (familial platelet disorder with predisposition to myeloid leukemia, FPDMM). Seminars in Hematology. 2017;**54**(2):75-80. DOI: 10.1053/j.seminhematol.2017.04.006

[6] Hayashi Y, Harada Y, Huang G, Harada H. Myeloid neoplasms with germ line *RUNX1* mutation. International Journal of Hematology. 2017;**106**(2):183-188. DOI: 10.1007/ s12185-017-2258-5

[7] Osato M, Asou N, Abdalla E, Hoshino K, Yamasaki H, Okubo T, et al. Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias. Blood. 1999;**93**(6):1817-1824

[8] Song WJ, Sullivan MG, Legare RD, Hutchings S, Tan X, Kufrin D, et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nature

Genetics. 1999;**23**(2):166-175. DOI: 10.1038/13793

[9] Churpek JE, Godley LA. How I diagnose and manage individuals at risk for inherited myeloid malignancies. Blood. 2016;**128**(14):1800-1813. DOI: 10.1182/blood-2016-05-670240

[10] Latger-Cannard V, Philippe C, Bouquet A, et al. Haematological spectrum and genotype-phenotype correlations in nine unrelated families with RUNX1 mutations from the French network on inherited platelet disorders. Orphanet Journal of Rare Diseases. 2016;**11**:49. DOI: 10.1186/ s13023-016-0432-0

[11] Braddock SR, South ST, Schiffman JD, Longhurst M, Rowe LR, Carey JC. Braddock-Carey syndrome: A 21q22 contiguous gene syndrome encompassing RUNX1. American Journal of Medical Genetics. Part A. 2016;**170**(10):2580-2586. DOI: 10.1002/ ajmg.a.37870

[12] Ripperger T, Tauscher M, Thomay K, et al. No evidence for ITSN1 loss in a patient with mental retardation and complex chromosomalr earrangements of 21q21-21q22. Leukemia Research 2013;**37**(6):721-723. DOI: 10.1016/j. leukres.2013.02.013

[13] Churpek JE, Lorenz R, Nedumgottil S, et al. Proposal for the clinical detection and management of patients and their family members with familial myelodysplastic syndrome/acute leukemia predisposition syndromes. Leukemia & Lymphoma. 2013;**54**(1):28-35. DOI: 10.3109/10428194.2012.701738

[14] Kozubík KS, Radová L, Pešová M, Réblová K, Trizuljak J, Plevová K, et al. C-terminal RUNX1 mutation in familial platelet disorder with predisposition to myeloid malignancies. International Journal

**47**

*Familial Leukemia Associated with Thrombocytopenia DOI: http://dx.doi.org/10.5772/intechopen.85303*

> underlying platelet function defect in a pedigree with familial platelet disorder with a predisposition to acute myelogenous leukemia: Potential role for candidate RUNX1 targets. Journal of Thrombosis and Haemostasis. 2014;**12**(5):761-772. DOI:

[22] Heller PG, Glembotsky AC, Gandhi MJ, et al. Low Mpl receptor expression in a pedigree with familial platelet disorder with predisposition to acute myelogenous leukemia and a novel AML1 mutation. Blood.

2005;**105**(12):4664-4670. DOI: 10.1182/

[23] Kaur G, Jalagadugula G, Mao G, Rao AK. RUNX1/core binding factor A2 Regulates platelet 12-lipoxygenasegene

(ALOX12): Studies in human RUNX1 haplodeficiency. Blood. 2010;**115**:3128-3135. DOI: 10.1182/

[24] Antony-Debre I, Bluteau D, Itzykson R, et al. MYH10 protein expression in platelets as a biomarker of RUNX1 and FLI1 alterations. Blood. 2012;**120**(13):2719-2722. DOI: 10.1182/

[25] Ng KP, Hu Z, Ebrahem Q, Negrotto S, Lausen J, Saunthararajah Y. Runx1 deficiency permits granulocyte lineage commitment but impairs subsequent maturation. Oncogene. 2013;**2**:e78. DOI:

[26] Satoh Y, Matsumura I, Tanaka H, et al. C-terminal mutation of RUNX1 attenuates the DNA-damage repair response in hematopoietic stem cells. Leukemia. 2012;**26**(2):303-311. DOI:

[27] Kirito K, Mitsumori T, Nagashima T, et al. A novel inherited singlenucleotide mutation in50-UTR in the transcription factor RUNX1 in familial platelet disorder with propensity to develop myeloid malignancies. Blood 2006;**108**:1917. http://www. bloodjournal.org/content/108/11/1917

blood-2009-04-214601

blood-2012-04-422352

10.1038/oncsis.2013.41

10.1038/leu.2011.202

10.1111/jth.12550

blood-2005-01-0050

of Hematology. 2018;**108**(6):652-657. DOI: 10.1007/s12185-018-2514-3. Epub

[15] Babushok DV, Bessler M. Genetic predisposition syndromes: When should they be considered in the work-up of MDS? Best Practice & Research. Clinical Haematology. 2015;**28**(1):55-68. DOI:

[16] Owen C, Barnett M, Fitzgibbon J. Familial myelodysplasia and acute myeloid leukaemia—A review. British Journal of Haematology.

10.1111/j.1365-2141.2007.06909.x

[17] Ripperger T, Tawana K, Kratz C, Schlegelberger B, Fitzgibbon J, Steinemann D. Clinical utility gene card for: Familial platelet disorder with associated myeloid malignancies.

European Journal of Human

blood-2008-07-168260

[20] Bluteau D, Glembotsky AC, Raimbault A, et al. Dysmegakaryopoiesis of FPD/ AML pedigrees with constitutional RUNX1 mutations is linked to myosin II deregulated expression. Blood. 2012;**120**(13):2708-2718. DOI: 10.1182/

blood-2012-04-422337

[21] Glembotsky AC, Bluteau D, Espasandin YR, et al. Mechanisms

ejhg.2015.278

Genetics. 2016;**24**:1232. DOI: 10.1038/

[18] Preudhomme C, Renneville A, Bourdon V, et al. High frequency of RUNX1 biallelic alteration in acute myeloid leukemia secondary to familial platelet disorder. Blood. 2009;**113**(22):5583-5587. DOI: 10.1182/

[19] Noris P, Biino G, Pecci A, et al. Platelet diameters in inherited thrombocytopenias: Analysis of 376 patients with all known disorders. Blood. 2014;**124**(6):e4-e10. DOI: 10.1182/blood-2014-03-564328

10.1016/j.beha.2014.11.004

2008;**140**:123-132. DOI:

2018 Aug 6

*Familial Leukemia Associated with Thrombocytopenia DOI: http://dx.doi.org/10.5772/intechopen.85303*

of Hematology. 2018;**108**(6):652-657. DOI: 10.1007/s12185-018-2514-3. Epub 2018 Aug 6

[15] Babushok DV, Bessler M. Genetic predisposition syndromes: When should they be considered in the work-up of MDS? Best Practice & Research. Clinical Haematology. 2015;**28**(1):55-68. DOI: 10.1016/j.beha.2014.11.004

[16] Owen C, Barnett M, Fitzgibbon J. Familial myelodysplasia and acute myeloid leukaemia—A review. British Journal of Haematology. 2008;**140**:123-132. DOI: 10.1111/j.1365-2141.2007.06909.x

[17] Ripperger T, Tawana K, Kratz C, Schlegelberger B, Fitzgibbon J, Steinemann D. Clinical utility gene card for: Familial platelet disorder with associated myeloid malignancies. European Journal of Human Genetics. 2016;**24**:1232. DOI: 10.1038/ ejhg.2015.278

[18] Preudhomme C, Renneville A, Bourdon V, et al. High frequency of RUNX1 biallelic alteration in acute myeloid leukemia secondary to familial platelet disorder. Blood. 2009;**113**(22):5583-5587. DOI: 10.1182/ blood-2008-07-168260

[19] Noris P, Biino G, Pecci A, et al. Platelet diameters in inherited thrombocytopenias: Analysis of 376 patients with all known disorders. Blood. 2014;**124**(6):e4-e10. DOI: 10.1182/blood-2014-03-564328

[20] Bluteau D, Glembotsky AC, Raimbault A, et al. Dysmegakaryopoiesis of FPD/ AML pedigrees with constitutional RUNX1 mutations is linked to myosin II deregulated expression. Blood. 2012;**120**(13):2708-2718. DOI: 10.1182/ blood-2012-04-422337

[21] Glembotsky AC, Bluteau D, Espasandin YR, et al. Mechanisms underlying platelet function defect in a pedigree with familial platelet disorder with a predisposition to acute myelogenous leukemia: Potential role for candidate RUNX1 targets. Journal of Thrombosis and Haemostasis. 2014;**12**(5):761-772. DOI: 10.1111/jth.12550

[22] Heller PG, Glembotsky AC, Gandhi MJ, et al. Low Mpl receptor expression in a pedigree with familial platelet disorder with predisposition to acute myelogenous leukemia and a novel AML1 mutation. Blood. 2005;**105**(12):4664-4670. DOI: 10.1182/ blood-2005-01-0050

[23] Kaur G, Jalagadugula G, Mao G, Rao AK. RUNX1/core binding factor A2 Regulates platelet 12-lipoxygenasegene (ALOX12): Studies in human RUNX1 haplodeficiency. Blood. 2010;**115**:3128-3135. DOI: 10.1182/ blood-2009-04-214601

[24] Antony-Debre I, Bluteau D, Itzykson R, et al. MYH10 protein expression in platelets as a biomarker of RUNX1 and FLI1 alterations. Blood. 2012;**120**(13):2719-2722. DOI: 10.1182/ blood-2012-04-422352

[25] Ng KP, Hu Z, Ebrahem Q, Negrotto S, Lausen J, Saunthararajah Y. Runx1 deficiency permits granulocyte lineage commitment but impairs subsequent maturation. Oncogene. 2013;**2**:e78. DOI: 10.1038/oncsis.2013.41

[26] Satoh Y, Matsumura I, Tanaka H, et al. C-terminal mutation of RUNX1 attenuates the DNA-damage repair response in hematopoietic stem cells. Leukemia. 2012;**26**(2):303-311. DOI: 10.1038/leu.2011.202

[27] Kirito K, Mitsumori T, Nagashima T, et al. A novel inherited singlenucleotide mutation in50-UTR in the transcription factor RUNX1 in familial platelet disorder with propensity to develop myeloid malignancies. Blood 2006;**108**:1917. http://www. bloodjournal.org/content/108/11/1917

**46**

*Germ Line Mutations Associated Leukemia*

[1] Fanconi G. Familiäre infantile perniziosaartige Anämie (perniziöses Blutbild und Konstitution). Jahrbuch

Genetics. 1999;**23**(2):166-175. DOI:

[9] Churpek JE, Godley LA. How I diagnose and manage individuals at risk for inherited myeloid malignancies. Blood. 2016;**128**(14):1800-1813. DOI: 10.1182/blood-2016-05-670240

[10] Latger-Cannard V, Philippe C, Bouquet A, et al. Haematological spectrum and genotype-phenotype correlations in nine unrelated families with RUNX1 mutations from the French network on inherited platelet disorders. Orphanet Journal of Rare Diseases. 2016;**11**:49. DOI: 10.1186/

[11] Braddock SR, South ST, Schiffman JD, Longhurst M, Rowe LR, Carey JC. Braddock-Carey syndrome: A 21q22 contiguous gene syndrome encompassing RUNX1. American Journal of Medical Genetics. Part A. 2016;**170**(10):2580-2586. DOI: 10.1002/

[12] Ripperger T, Tauscher M, Thomay K, et al. No evidence for ITSN1 loss in a patient with mental retardation and complex chromosomalr earrangements of 21q21-21q22. Leukemia Research 2013;**37**(6):721-723. DOI: 10.1016/j.

[13] Churpek JE, Lorenz R, Nedumgottil

S, et al. Proposal for the clinical detection and management of patients and their family members with familial myelodysplastic syndrome/acute leukemia predisposition syndromes. Leukemia &

Lymphoma. 2013;**54**(1):28-35. DOI: 10.3109/10428194.2012.701738

[14] Kozubík KS, Radová L, Pešová M, Réblová K, Trizuljak J, Plevová K, et al. C-terminal RUNX1

mutation in familial platelet disorder with predisposition to myeloid malignancies. International Journal

10.1038/13793

s13023-016-0432-0

ajmg.a.37870

leukres.2013.02.013

Kinderheilk. 1927;**117**:257-280

[2] Wlodarski MW, Niemeyer CM. Introduction: Genetic

Genetics. 2016;**89**(2):141-153

[5] Schlegelberger B, Heller PG. *RUNX1* deficiency (familial platelet disorder with predisposition to myeloid leukemia, FPDMM). Seminars in Hematology. 2017;**54**(2):75-80. DOI: 10.1053/j.seminhematol.2017.04.006

[6] Hayashi Y, Harada Y, Huang G, Harada H. Myeloid neoplasms with germ line *RUNX1* mutation. International Journal of Hematology. 2017;**106**(2):183-188. DOI: 10.1007/

[7] Osato M, Asou N, Abdalla E, Hoshino K, Yamasaki H, Okubo T, et al. Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias. Blood.

[8] Song WJ, Sullivan MG, Legare RD, Hutchings S, Tan X, Kufrin D, et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nature

s12185-017-2258-5

1999;**93**(6):1817-1824

syndromes predisposing to myeloid neoplasia. Seminars in Hematology. 2017;**54**(2):57-59. DOI: 10.1053/j. seminhematol.2017.05.001

[3] Pecci A. Diagnosis and treatment of inherited thrombocytopenias. Clinical

[4] Arber DA, Orazi A, Hasserijan R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;**127**(20):2391-2405. DOI: 10.1182/blood-2016-03-643544

**References**

[28] Antony-Debre I, Manchev VT, Balayn N, et al. Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia. Blood. 2015;**125**(6):930-940. DOI: 10.1182/ blood-2014-06-585513

[29] Ripperger T, Tauscher M, Ehlert L, et al. Childhood onset of leukaemia in familial platelet disorder with propensity for myeloid malignancies due to an intragenic RUNX1 deletion. Haematologica. 2012;**97**:s3-S14

[30] Churpek JE, Pyrtel K, Kanchi KL, et al. Genomic analysis of germline and somatic variants in familial myelodysplasia/acute myeloid leukemia. Blood. 2015;**126**:2484-2490. DOI: 10.1182/blood-2015-04-641100

[31] Sakurai M, Kasahara H, Yoshida K, et al. Genetic basis of myeloid transformation in familial platelet disorder/acute myeloid leukemia patients with haploinsufficient RUNX1 allele. Blood Cancer Journal. 2016;**6**:e392. DOI: 10.1002/gcc.21918

[32] Haferlach C, Bacher U, Schnittger S, et al. ETV6 rearrangements are recurrent in myeloid malignancies and are frequently associated with other genetic events. Genes, Chromosomes & Cancer. 2012;**51**:328-337. DOI: 10.1002/ gcc.21918

[33] Yoshimi A, ToyaT KM, et al. Recurrent CDC25C mutations drive malignant transformation in FPD/ AML. Nature Communications. 2014;**5**:4770. DOI: 10.1038/ncomms5770

[34] Haslam K, Langabeer SE, Hayat A, Conneally E, Vandenberghe E. Targeted next-generation sequencing of familial platelet disorder with predisposition to acute myeloid leukaemia. British Journal of Haematology. 2016;**175**:161-163. DOI: 10.1111/bjh.13838

[35] Golub TR, Barker GF, Lovett M, Gilliland DG. Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell. 1994;**77**(2):307-316

[36] de Braekeleer E, Auffret R, Garcia JR, Padilla JM, Fletes CC, Morel F, et al. Identification of NIPBL, a new ETV6 partner gene in t(5;12) (p13;p13)-associated acute megakaryoblastic leukemia. Leukemia & Lymphoma. 2013;**54**(2):423-424. DOI: 10.3109/10428194.2012.706288

[37] Hollenhorst PC, McIntosh LP, Graves BJ. Genomic and biochemical insights into the specificity of ETS transcription factors. Annual Review of Biochemistry. 2011;**80**:437-471. DOI: 10.1146/annurev.biochem.79. 081507.103945

[38] De Braekeleer E, Douet-Guilbert N, Morel F, Le Bris MJ, Basinko A, De Braekeleer M. ETV6 fusion genes in hematological malignancies: A review. Leukemia Research. 2012;**36**(8):945-961. DOI: 10.1016/j.leukres.2012.04.010

[39] Papaemmanuil E. RapadoI, Li Y, et al. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia. Nature Genetics. 2014;**46**(2):116-125. DOI: 10.1038/ng.2874

[40] van Doorn SB, Spensberger D, de Knegt Y, Tang M, Spensberger D, et al. Somatic heterozygous mutations in ETV6 (TEL) and frequent absence of ETV6 protein in acute myeloid leukemia. Oncogene. 2005;**24**(25): 4129-4137. DOI: 10.1038/sj.onc.1208588

[41] Van Vlierberghe P, Ambesi-Impiombato A, Perez-Garcia A, et al. ETV6 mutations in early immature human T cell leukemias. The Journal of Experimental Medicine. 2011;**208**(13):2571-2579. DOI: 10.1084/ jem.20112239

**49**

*Familial Leukemia Associated with Thrombocytopenia DOI: http://dx.doi.org/10.5772/intechopen.85303*

> [49] Moriyama T, Metzger ML, Wu G, et al. Germline genetic variation in ETV6 and risk of childhood acute lymphoblastic leukaemia: A systematic genetic study. The Lancet Oncology. 2015;**16**(16):1659-1666. DOI: 10.1016/

> [50] Topka S, Vijai J, Walsh MF, et al. Germline ETV6 mutations confer susceptibility to acute lymphoblastic leukemia and thrombocytopenia. PLoS Genetics. 2015;**11**(6):e1005262. DOI: 10.1371/journal.pgen.1005262

Sobieszczuk P, Wasylyk B. Reversion of Ras transformed cells by ETS transdominant mutants. Oncogene.

[52] Fenrick R, Wang L, Nip J, et al. TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1. Molecular and Cellular Biology.

S1470-2045(15)00369-1

[51] Wasylyk C, Maira SM,

1994;**9**(12):3665-3673

2000;**20**(16):5828-5839

[53] Park H, Seo Y, Kim JI, Kim WJ, Choe SY. Identification of the nuclear localization motif in the ETV6 (TEL) protein. Cancer Genetics and Cytogenetics. 2006;**167**(2):117-121. DOI: 10.1016/j.cancergencyto.2006.01.006

[54] Wang LC, Kuo F, Fujiwara Y, Gilliland DG, Golub TR, Orkin SH. Yolk

sac angiogenic defect and intraembryonic apoptosis in mice lacking the Ets-related factor TEL. The EMBO Journal. 1997;**16**(14):4374-4383. DOI:

[55] Hock H, Meade E, Medeiros S, et al. Tel/Etv6 is an essential and selective regulator of adult hematopoietic stem cell survival. Genes & Development. 2004;**18**(19):2336-2341. DOI: 10.1101/

[56] Wang LC, Swat W, Fujiwara Y, et al. The TEL/ETV6 gene is required

10.1093/emboj/16.14.4374

gad.1239604

[42] Zhang J, Ding L, Holmfeldt L, et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature. 2012;**481**(7380): 157-163. DOI: 10.1038/nature10725

[43] Zhang J, Mullighan CG, Harvey RC, et al. Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: A report from the children's oncology group. Blood. 2011;**118**(11):3080-3087. DOI: 10.1182/

[44] Lohr JG, Stojanov P, Lawrence MS, et al. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(10): 3879-3884. DOI: 10.1073/

[45] Zhang MY, Churpek JE, Keel SB, et al. Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy. Nature Genetics. 2015;**47**(2).

[46] Noetzli L, Lo RW, Lee-Sherick AB, et al. Germline mutations inETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia. Nature Genetics. 2015;**47**(5):535-538. DOI:

[47] Melazzini F, Palombo F, Balduini A, Doubek M, et al. Clinical and pathogenic features of ETV6-related thrombocytopenia with predisposition to acute lymphoblastic leukemia. Haematologica. 2016;**101**(11):1333-

[48] Poggi M, Canault M, Favier M, et al. Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors. Haematologica. 2017;**102**(2):282-294. DOI: 10.3324/haematol.2016.147694

blood-2011-03-341412

pnas.1121343109

DOI: 10.1038/ng.3177

10.1038/ng.3253

1342. 0-5. DOI: 10.3324/ haematol.2016.147496

*Familial Leukemia Associated with Thrombocytopenia DOI: http://dx.doi.org/10.5772/intechopen.85303*

[42] Zhang J, Ding L, Holmfeldt L, et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature. 2012;**481**(7380): 157-163. DOI: 10.1038/nature10725

*Germ Line Mutations Associated Leukemia*

[28] Antony-Debre I, Manchev VT, Balayn N, et al. Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia. Blood. 2015;**125**(6):930-940. DOI: 10.1182/

beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation.

[36] de Braekeleer E, Auffret R, Garcia JR, Padilla JM, Fletes CC, Morel F, et al. Identification of NIPBL, a new ETV6 partner gene in t(5;12) (p13;p13)-associated acute megakaryoblastic leukemia. Leukemia & Lymphoma. 2013;**54**(2):423-424. DOI: 10.3109/10428194.2012.706288

[37] Hollenhorst PC, McIntosh LP, Graves BJ. Genomic and biochemical insights into the specificity of ETS transcription factors. Annual Review of Biochemistry. 2011;**80**:437-471. DOI: 10.1146/annurev.biochem.79.

[38] De Braekeleer E, Douet-Guilbert N, Morel F, Le Bris MJ, Basinko A, De Braekeleer M. ETV6 fusion genes in hematological malignancies: A review. Leukemia Research. 2012;**36**(8):945-961. DOI: 10.1016/j.leukres.2012.04.010

[39] Papaemmanuil E. RapadoI, Li Y, et al. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia. Nature Genetics. 2014;**46**(2):116-125. DOI:

[40] van Doorn SB, Spensberger D, de Knegt Y, Tang M, Spensberger D, et al. Somatic heterozygous mutations in ETV6 (TEL) and frequent absence of ETV6 protein in acute myeloid leukemia. Oncogene. 2005;**24**(25): 4129-4137. DOI: 10.1038/sj.onc.1208588

[41] Van Vlierberghe P, Ambesi-Impiombato A, Perez-Garcia A, et al. ETV6 mutations in early immature human T cell leukemias. The Journal of Experimental Medicine. 2011;**208**(13):2571-2579. DOI: 10.1084/

081507.103945

10.1038/ng.2874

jem.20112239

Cell. 1994;**77**(2):307-316

[29] Ripperger T, Tauscher M, Ehlert L, et al. Childhood onset of leukaemia in familial platelet disorder with propensity for myeloid malignancies due to an intragenic RUNX1 deletion. Haematologica. 2012;**97**:s3-S14

[30] Churpek JE, Pyrtel K, Kanchi KL, et al. Genomic analysis of germline and somatic variants in familial

myelodysplasia/acute myeloid leukemia. Blood. 2015;**126**:2484-2490. DOI: 10.1182/blood-2015-04-641100

[31] Sakurai M, Kasahara H, Yoshida K, et al. Genetic basis of myeloid transformation in familial platelet disorder/acute myeloid leukemia patients with haploinsufficient RUNX1 allele. Blood Cancer Journal. 2016;**6**:e392. DOI: 10.1002/gcc.21918

[32] Haferlach C, Bacher U, Schnittger S, et al. ETV6 rearrangements are recurrent in myeloid malignancies and are frequently associated with other genetic events. Genes, Chromosomes & Cancer. 2012;**51**:328-337. DOI: 10.1002/

[33] Yoshimi A, ToyaT KM, et al. Recurrent CDC25C mutations drive malignant transformation in FPD/ AML. Nature Communications.

2014;**5**:4770. DOI: 10.1038/ncomms5770

[34] Haslam K, Langabeer SE, Hayat A, Conneally E, Vandenberghe E. Targeted next-generation sequencing of familial platelet disorder with predisposition to acute myeloid leukaemia. British Journal of Haematology. 2016;**175**:161-163. DOI:

[35] Golub TR, Barker GF, Lovett M, Gilliland DG. Fusion of PDGF receptor

blood-2014-06-585513

**48**

10.1111/bjh.13838

gcc.21918

[43] Zhang J, Mullighan CG, Harvey RC, et al. Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: A report from the children's oncology group. Blood. 2011;**118**(11):3080-3087. DOI: 10.1182/ blood-2011-03-341412

[44] Lohr JG, Stojanov P, Lawrence MS, et al. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(10): 3879-3884. DOI: 10.1073/ pnas.1121343109

[45] Zhang MY, Churpek JE, Keel SB, et al. Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy. Nature Genetics. 2015;**47**(2). DOI: 10.1038/ng.3177

[46] Noetzli L, Lo RW, Lee-Sherick AB, et al. Germline mutations inETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia. Nature Genetics. 2015;**47**(5):535-538. DOI: 10.1038/ng.3253

[47] Melazzini F, Palombo F, Balduini A, Doubek M, et al. Clinical and pathogenic features of ETV6-related thrombocytopenia with predisposition to acute lymphoblastic leukemia. Haematologica. 2016;**101**(11):1333- 1342. 0-5. DOI: 10.3324/ haematol.2016.147496

[48] Poggi M, Canault M, Favier M, et al. Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors. Haematologica. 2017;**102**(2):282-294. DOI: 10.3324/haematol.2016.147694

[49] Moriyama T, Metzger ML, Wu G, et al. Germline genetic variation in ETV6 and risk of childhood acute lymphoblastic leukaemia: A systematic genetic study. The Lancet Oncology. 2015;**16**(16):1659-1666. DOI: 10.1016/ S1470-2045(15)00369-1

[50] Topka S, Vijai J, Walsh MF, et al. Germline ETV6 mutations confer susceptibility to acute lymphoblastic leukemia and thrombocytopenia. PLoS Genetics. 2015;**11**(6):e1005262. DOI: 10.1371/journal.pgen.1005262

[51] Wasylyk C, Maira SM, Sobieszczuk P, Wasylyk B. Reversion of Ras transformed cells by ETS transdominant mutants. Oncogene. 1994;**9**(12):3665-3673

[52] Fenrick R, Wang L, Nip J, et al. TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1. Molecular and Cellular Biology. 2000;**20**(16):5828-5839

[53] Park H, Seo Y, Kim JI, Kim WJ, Choe SY. Identification of the nuclear localization motif in the ETV6 (TEL) protein. Cancer Genetics and Cytogenetics. 2006;**167**(2):117-121. DOI: 10.1016/j.cancergencyto.2006.01.006

[54] Wang LC, Kuo F, Fujiwara Y, Gilliland DG, Golub TR, Orkin SH. Yolk sac angiogenic defect and intraembryonic apoptosis in mice lacking the Ets-related factor TEL. The EMBO Journal. 1997;**16**(14):4374-4383. DOI: 10.1093/emboj/16.14.4374

[55] Hock H, Meade E, Medeiros S, et al. Tel/Etv6 is an essential and selective regulator of adult hematopoietic stem cell survival. Genes & Development. 2004;**18**(19):2336-2341. DOI: 10.1101/ gad.1239604

[56] Wang LC, Swat W, Fujiwara Y, et al. The TEL/ETV6 gene is required specifically for hematopoiesis in the bone marrow. Genes & Development. 1998;**12**(15):2392-2402. DOI: 10.1101/ gad.12.15.2392

[57] Noris P, Favier R, Alessi MC, Geddis AE, Kunishima S, Heller PG, et al. *ANKRD26*-related thrombocytopenia and myeloid malignancies. Blood. 2013;**122**(11):1987-1989. DOI: 10.1182/ blood-2013-04-499319

[58] Savoia A, Del Vecchio M, Totaro A, Perrotta S, Amendola G, Moretti A, et al. An autosomal dominant thrombocytopenia gene maps to chromosomal region 10p. American Journal of Human Genetics. 1999;**65**:1401-1405. DOI: 10.1086/302637

[59] Drachman JG, Jarvik GP, Mehaffey MG. Autosomal dominant thrombocytopenia: Incomplete megakaryocyte differentiation and linkage to human chromosome 10. Blood. 2000;**96**:118-125

[60] Pippucci T, Savoia A, Perrotta S, et al. Mutations in the 5' UTR of ANKRD26, the ankirin repeat domain 26 gene, cause an autosomal-dominant form of inherited thrombocytopenia, THC2. American Journal of Human Genetics. 2011;**88**(1):115-120. DOI: 10.1016/j.ajhg.2010.12.006

[61] Noris P, Perrotta S, Seri M, et al. Mutations in ANKRD26 are responsible for a frequent form of inherited thrombocytopenia: Analysis of 78 patients from 21 families. Blood. 2011;**117**(24):6673-6680. DOI: 10.1182/ blood-2011-02-336537

[62] Hahn Y, Bera TK, Pastan IH, Lee B. Duplication and extensive remodeling shaped POTE family genes encoding proteins containing ankyrin repeat and coiled coil domains. Gene. 2006;**366**:238-245. DOI: 10.1016/j. gene.2005.07.045

[63] Macaulay IC, Tijssen MR, Thijssen-Timmer DC, Gusnanto A, Steward M, Burns P, et al. Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins. Blood. 2007;**109**:3260-3269. DOI: 10.1182/blood-2006-07-036269

[64] Bera TK, Liu XF, Yamada M, Gavrilova O, Mezey E, Tessarollo L, et al. A model for obesity and gigantism due to disruption of the Ankrd26 gene. Proceedings of the National Academy of Sciences. 2008;**105**:270-275. DOI: 10.1073/pnas.0710978105

[65] Bluteau O et al. A landscape of germ line mutations in a cohort of inherited bone marrow failure patients. Blood; Feb 15 2018;**131**(7):717-732. DOI: 10.1182/blood-2017-09-806489

[66] Rheingold SR. Acute myeloid leukemia in a child with hereditary thrombocytopenia. Pediatric Blood & Cancer. 2007;**48**(1):105-107. DOI: 10.1002/pbc.20677

[67] Yu M, Wang J, Zhu Z, et al. Prognostic impact of MYH9 expression on patients with acute myeloid leukemia. Oncotarget. 2016;**8**(1):156-163. DOI: 10.18632/ oncotarget.10613

**51**

Section 4

Energy Failure and

Leukemia

Section 4
